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Phages targeting infected tissues: novel approach to phage therapy Andrzej Górski*,1,2, Krystyna Dąbrowska1, Katarzyna Hodyra-Stefaniak1, Jan Borysowski2, Ryszard Międzybrodzki1,2 & Beata Weber-Dąbrowska1
ABSTRACT While the true efficacy of phage therapy still requires formal confirmation in clinical trials, it continues to offer realistic potential treatment in patients in whom antibiotics have failed. Novel developments and approaches are therefore needed to ascertain that future clinical trials would evaluate the therapy in its optimal form thus allowing for reliable conclusions regarding the true value of phage therapy. In this article, we present our vision to develop and establish a bank of phages specific to most threatening pathogens and armed with homing peptides enabling their localization in infected tissues in densities assuring efficient and stable eradication of infection. A key factor of successful treatment of infection is sufficient exposure of the antimicrobial agent at the site of bacterial invasion, in most cases this is the interstitial space of infected organs. Antibiotics must not only penetrate the infected site, but also reach sufficient concentrations in situ [1,2] . In some clinical settings effective concentrations of antibiotics are difficult to achieve by systemic application because of poor blood circulation to burns [3] , bones, diabetic ulcers [4,5] and the prostate gland [6] . Bacteriophages (phages) are viruses of bacteria that can replicate using lytic cycle (ending in bacterial cell destruction) or lysogenic cycle (when phages integrate with the bacterial genome). The growing problem of antibiotic resistance has greatly revived interest in phage therapy (which uses lytic phages only) [7–12] . The clinical efficacy of the therapy still requires confirmation in clinical trials, although the results of a small POC CT are encouraging [13] , yet the observations from experimental therapy offered to patients in whom antibiotic therapy had failed strongly suggest that it may bring improvement in some 40% of cases and eradication of infection/recovery in almost 20% of them [14] . These data, of course, should be interpreted with caution as they are derived from observational studies and not clinical trials. However, such studies also have value in detecting signals about the benefits and risks, and help formulate hypotheses to be tested in subsequent trials [15] . The relatively short life of phages is a very important problem in phage therapy. Therefore, some experimental attempts were made to obtain phage mutants with improved pharmacokinetic features. Merril et al. described a mutant with a minor modification in its capsid protein that was able to escape the reticuloendothelial system (RES) and, therefore, persisted in the circulation with improved therapeutic potential [16] . Similar results were obtained by Capparelli et al. who found that a Staphylococcus aureus phage mutant could persist in the circulation for 21–25 days (wild phage up to 2 days) and, interestingly, did not induce the formation of n eutralizing a ntibodies [17] .
KEYWORDS
• antibiotic resistance • homing peptides • phage display • phage therapy
L Hirszfeld Institute of Immunology & Experimental Therapy, Polish Academy of Science, Wroclaw, Poland Department of Clinical Immunology, Medical University of Warsaw, Warsaw, Poland *Author for correspondence: [email protected] 1 2
10.2217/FMB.14.126 © 2015 Future Medicine Ltd
Future Microbiol. (2015) 10(2), 199–204
part of
ISSN 1746-0913
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Perspective Górski, Dąbrowska, Hodyra-Stefaniak et al. The correlation between the ability of the mutant phage to escape capture by the RES and the ability to rescue bacteremic mice suggests that such a phenomenon may improve the efficacy of phage therapy [16] . However, it is unclear whether this approach might also be useful in treating localized infections where vascular exit and parenchymal spread are necessary. In addition, it is unclear whether long persisting phagemia do not cause side effects (e.g., changes in immune reactivity and hematologic system, among others), which could adversely influence the final effect of therapy. The spread of phages throughout the organism following their intravenous administration (and using other routes) has been studied in some detail in mice and was summarized by Dabrowska et al. [18] . In particular, while significant titers of phages were present in the lung, kidney, spleen and liver, they were approximately 100-times lower than in the blood. In Matsuzaki’s experiments, following intraperitoneal (ip.) administration, phage can be detected in these organs in concentrations of 10–106 PFU/g [19] . When given orally to mice and human volunteers, even without an antacid, doses as low as 103 PFU/ml led to detection of phage in the feces, suggesting that phage can cross the stomach barrier [20] . What is more, phage concentration in a particular organ also depends on the absence or presence of susceptible bacteria. For example, phages may persist in infected fish spleen up to 5 days after ip. administration but only up to 24 h in the spleen of noninfected fish, which possibly reflects phage multiplication in situ [21] . The pharmacology of phage therapy has been analyzed and discussed by Levin and Bull [22] , and Payne and Janse [23,24] . Recently, Abedon has offered another in-depth analysis that introduced the term ‘inundation threshold’ to indicate the minimum phage density that can keep a bacterial infection from getting worse and, precisely, the density that keeps a bacterial culture from becoming more concentrated [25] . This is equivalent to the density that allows for bacterial killing in equilibrium with bacterial replication. According to the author’s calculations, the inundation threshold is equal to at least 107 phages/ml, but for a vigorously growing infection higher densities may be necessary. A more practical measure of phage ability to counteract infection is the killing titer (the density required to clear bacteria),
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which – according to Abedon – should be at least 108 phages/ml; achieving such phage densities over the course of treatment should be the dosing goal in phage therapy protocols [25] . Consequently, an important goal of optimal phage therapy protocol is to apply phages more directly to infections [25] . As stated, phage therapy efficiency is highly dependent on attaining a high phage ‘killing titer.’ If bacteria infiltrate to locations which phages are less able to penetrate, phage densities resulting from therapeutic administration may not be sufficient to achieve bacterial eradication. Thus, self amplification of phage in situ may be a mechanism enabling the therapeutic effect. However, it is unclear whether phage replication may always take place in infected organs at least to boost phage numbers to sufficient levels causing efficient elimination of bacteria. Therefore, as pointed out by Abedon, sufficiently high or repeated phage doses should be administered, rather than expecting that phage reproduce in situ, to achieve therapy success [25,26] . This effect should be achieved by a slow intravenous drip of a highly concentrated phage preparation. Notably, using of anion-exchange chromatography phages can be purified and concentrated to 1013 PFU/ml [27,28] . Selective targeting of a systematically administered drug would enhance the efficacy of treatment, an approach that could be of special significance for improving the success of antimicrobial treatment, including phage therapy. With this in mind, we envisage a novel approach to phage therapy based on bacteriophages presenting organ-specific peptides targeting them to infected organs, which should significantly enhance the efficacy of the therapy, reduce its potential side effects and protect against the neutralizing action of the immune system (e.g., production of phageinactivating antibodies and phagocytosis by neutrophils/monocytes among others). Phage can also induce opsonins that may restore their vulnerability to the RES with subsequent decay; moreover, possible toxicity of phage–antibody complexes should also be considered [29] . The renal glomerulus is particularly predisposed to the deposition of antigen–antibody complexes, which may cause both acute and chronic glomerular injury with serum sickness, acute and chronic glomerulonephritis [30] . Notably, virus–antibody complexes may mediate vasculitis of different tissues including skin, k idney, peripheral and CNS and liver [31] .
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Phages targeting infected tissues: novel approach to phage therapy As shown by the Benhar group, phages can be targeted to specific bacteria: targeted phages chemically conjugated with antibiotics may be nontoxic and less immunogenic as a result of drug conjugation [32–34] . However, this interesting approach, which offers selectivity of antibiotic action, does not solve the problem of antibiotic resistance. The fundamental studies of the Ruoslahti group have revealed that the endothelia of vessels of different organs are molecularly different (vascular zip codes). Those studies used in vivo phage display: a library of phage displaying random peptides is injected systematically into mice, followed by removal of target organs, amplification of the bound phage and subjecting the amplified pool to another round of selection, with positive selection at the target tissue. Initially, the group has identified homing peptides for brain and kidney [35] ; subsequent work has allowed for identification of other organ-specific peptides by the use of phage libraries [36] . Such phage nanoparticles armed with tissue homing peptides can ensure the advantage of topical application of drugs: high local concentration at the site of disease process and low systemic exposure (referred to as synaptic drug delivery) [37] . Parenchymal cells frequently express the same marker as endothelium; moreover, peptides enhancing nanoparticle transport from endothelium to extravascular tissue have been discovered and shown to facilitate nanoparticle penetration of tissues. Importantly, this system allowing phage extravasation and tissue penetration can be activated in a tissue-specific manner [37] . Use of such system could be very important in view of some experimental data suggesting inadequate capacity of unmodified phages to get out of the bloodstream and penetrate tissues [25] . Thus, the available data suggest that methods aiming at improved transport of phages to infected tissue may enhance the efficacy of phage therapy. At least three lines of evidence support this assumption. First, our knowledge about phage penetration to tissues following administration by routes other than topical is limited (almost nonexistent in man) and the available data are discordant. The prospects for acquiring such data in man in the near future look rather gloomy: lack of funding to carry out clinical trials and preclinical studies including pharmacokinetics in man constitute significant obstacles for progress in this area. The pharma industry is reluctant to fund such
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Perspective
studies in view of the lack of a specific frame for phage therapy under the current Medicinal Product Regulation. Also, in the United States, the amount of research and testing required by the US FDA is hampering the resurgence of phage therapy [38] . Second, the available data do not suggest that any route of administration could ascertain that tissue density of phages may reach the values suggested by Abedon to assure efficient local elimination of bacteria. Third, two ongoing clinical trials (one funded by the EC and the other by Nestle), as far as we are aware, do not include detailed pharmacokinetic studies [39,40] . On the other hand, recent decisions of the FDA facilitating access to experimental therapy ‘expanded access’ [41] and the increasing problem of microbial antibiotic resistance strongly suggest that phage therapy will continue to have a place in the treatment of patients in whom no other treatment is available and could find a much wider application – provided clinical trials confirm its efficacy. Many phages are polyvalent and infect members of several bacterial genera with phylogenetic relationship, which is relevant for phage therapy [42] . Both polyvalent with a broad host range and monovalent phages are used in therapy. At our center, it is possible to find an active specific phage for 90% of S. aureus, 72% of Pseudomonas aeruginosa and 77% of Enterococcus faecalis strains [14] . From the data presented above, it seems that phages presenting homing peptides (applied alone or perhaps along with transport-enhancing peptides) should allow for attaining higher local tissue concentrations of phages than those achievable using wild phages, and may therefore be an interesting alternative to currently used therapy, at least in infections where phage penetration could be reasonably expected to be poor (e.g., bone and prostate, among others). While there are no convincing data indicating that phage can cross the intestinal wall and migrate to blood or lymph in humans, a phenomenon known as bacterial translocation can also include phages [43] ; if this is indeed the case, oral administration of tissue-targeting phages might be a convenient and efficient novel form of phage therapy. The range of pathogens that can be eliminated by the targeted phages is directly related to the progress in phage engineering. Availability of molecular systems for phage modifications is the main limitation here. Currently, coliphages can be easily modified due to the abundance of Escherichia coli laboratory strains, coli-related vectors and
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Perspective Górski, Dąbrowska, Hodyra-Stefaniak et al. well established systems for phage display. Nevertheless, one may foresee upcoming solutions for constructing targeting phages against virtually any bacteria: synthetic genomics [44] will enable unlimited specificity of targeted phages. The idea of tissue-specific engineered phages is strongly supported by recent development of methodologies for phage engineering. ‘Classical’ phage display, based on introducing foreign gene sequences into phage genomes [45] , is not likely to offer a widely accepted therapeutic approach. This is because modified phages become genetically modified organisms. Environmental release of such constructs is strongly restricted and their use in general is not well accepted by the society. Some new types of phage display do not require introducing foreign DNA to phages. These can be in vitro modifications of mature phages as shown by Li et al. who reported the first presentation of anthrax toxin on bacteriophage T4 [46] . Other method is based on the competitive phage display: a phage propagates on a bacterial host that additionally to the phage produces also engineered fusions of phage proteins with active peptides or proteins. These fusions can be randomly incorporated into phage capsid, resulting in a modified phage particle without genetic manipulations in the phage [47,48] . Most currently used methods based on phage display use filamentous, lambda and T family phages, which ensure good peptide presentation while phage biological activity in infecting bacteria is maintained. In theory, other phages relevant for combating antibiotic-resistant infections could also be equipped with organ-specific homing receptors. However, the biological a ctivity of such constructs needs to be confirmed. Also chemical modifications, which do not employ phage display, can be used to anchor designed peptides on a phage surface. To date, this approach has been used mostly for nonprotein compounds [32,49] , but it could be further developed to modify phage surface with amino acidic motifs, for example, by dual conjugation of prosthetic groups. Our data indicating that phages can also mediate anti-inflammatory effects, which may be at least partly independent of their antibacterial action [50] , add another important dimension to this approach, as they suggest that organ-specific phages could mediate local anti-inflammatory action without significant side effects typical for currently used standard drugs (steroids and nonsteroidal anti-inflammatory agents). However,
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this promising effect needs to be validated by clinical trials. Conclusion In conclusion, the capacity of phages to leave the bloodstream, migrate to the infected tissues and achieve concentrations necessary to eradicate bacterial infection remains a significant challenge. This problem may be circumvented by constructing phages armed with tissue-specific homing peptides, which could be targeted to reach infected organs. This novel approach may significantly improve the effectiveness of phage therapy. Future perspective In the past decade, phage-expressing homing peptides have been shown to offer a novel approach to experimental therapy of a variety of disorders (cancer, atherosclerosis, tissue scarring). One could anticipate a similar pace of progress in the currently suggested field. Comparable time should allow for construction, experimental confirmation and perhaps initial clinical trials using phages targeted to penetrate bone and prostate, whose infection poses difficult medical challenges. The construction of phages specific for the most relevant pathogens and targeting organs and tissues could result in the establishment of a phage bank; the authors believe support for the costs of its operations should be sought from governmental or pharmaceutical company resources. Dedication This article is dedicated to the memory of Professor Ludwik Hirszfeld, founder of our Institute, who had contributed greatly to the development of phage therapy.
Financial & competing interests disclosure A Górski and B Weber-Dąbrowska are co-inventors on a patent covering phage preparations owned by the Institute. K Dąbrowska and A Górski are co-inventors on a patent application related to phage purification with the competitive phage display. This work was supported by National Science Centre in Poland (grant no. UMO-2014/13/N/ NZ6/03985) and by statutory funds of Medical University of Warsaw (1MG/N/2015). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
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Phages targeting infected tissues: novel approach to phage therapy
Perspective
EXECUTIVE SUMMARY ●●
Microbial antibiotic resistance constitutes one of the most challenging threats in medicine today. Given the slow rate of progress in the introduction of new drugs, phage therapy offers an interesting alternative to antibiotics; therefore, research and new developments in this area are urgently needed.
●●
The relatively short life of phages and their questionable tissue penetration warrant novel approaches enabling sufficient tissue density of phages to be attained to ensure effective eradication of infection.
●●
Phage display has been demonstrated to be an efficacious biotechnology tool yielding phages able to target specific
organs and tissues. Therefore, phage display could also be used to increase phage concentration in infected organs, which may further allow expansion of phage capability to reach specific pathogens that cause localized infections. This may be of special importance in case of tissues where penetration of antimicrobial agents is questionable. ●●
Regarding phage display, no data are available on the use of phages other than filamentous, lambda and T-family
phages; therefore, research is needed to confirm that such targeted phages also retain their efficient antibacterial activity. resistant bacteria. Adv. Exp. Med. Biol. 807, 97–110 (2014).
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Phages targeting infected tissues: novel approach to phage therapy Andrzej Górski*,1,2, Krystyna Dąbrowska1, Katarzyna Hodyra-Stefaniak1, Jan Borysowski2, Ryszard Międzybrodzki1,2 & Beata Weber-Dąbrowska1
ABSTRACT While the true efficacy of phage therapy still requires formal confirmation in clinical trials, it continues to offer realistic potential treatment in patients in whom antibiotics have failed. Novel developments and approaches are therefore needed to ascertain that future clinical trials would evaluate the therapy in its optimal form thus allowing for reliable conclusions regarding the true value of phage therapy. In this article, we present our vision to develop and establish a bank of phages specific to most threatening pathogens and armed with homing peptides enabling their localization in infected tissues in densities assuring efficient and stable eradication of infection. A key factor of successful treatment of infection is sufficient exposure of the antimicrobial agent at the site of bacterial invasion, in most cases this is the interstitial space of infected organs. Antibiotics must not only penetrate the infected site, but also reach sufficient concentrations in situ [1,2] . In some clinical settings effective concentrations of antibiotics are difficult to achieve by systemic application because of poor blood circulation to burns [3] , bones, diabetic ulcers [4,5] and the prostate gland [6] . Bacteriophages (phages) are viruses of bacteria that can replicate using lytic cycle (ending in bacterial cell destruction) or lysogenic cycle (when phages integrate with the bacterial genome). The growing problem of antibiotic resistance has greatly revived interest in phage therapy (which uses lytic phages only) [7–12] . The clinical efficacy of the therapy still requires confirmation in clinical trials, although the results of a small POC CT are encouraging [13] , yet the observations from experimental therapy offered to patients in whom antibiotic therapy had failed strongly suggest that it may bring improvement in some 40% of cases and eradication of infection/recovery in almost 20% of them [14] . These data, of course, should be interpreted with caution as they are derived from observational studies and not clinical trials. However, such studies also have value in detecting signals about the benefits and risks, and help formulate hypotheses to be tested in subsequent trials [15] . The relatively short life of phages is a very important problem in phage therapy. Therefore, some experimental attempts were made to obtain phage mutants with improved pharmacokinetic features. Merril et al. described a mutant with a minor modification in its capsid protein that was able to escape the reticuloendothelial system (RES) and, therefore, persisted in the circulation with improved therapeutic potential [16] . Similar results were obtained by Capparelli et al. who found that a Staphylococcus aureus phage mutant could persist in the circulation for 21–25 days (wild phage up to 2 days) and, interestingly, did not induce the formation of n eutralizing a ntibodies [17] .
KEYWORDS
• antibiotic resistance • homing peptides • phage display • phage therapy
L Hirszfeld Institute of Immunology & Experimental Therapy, Polish Academy of Science, Wroclaw, Poland Department of Clinical Immunology, Medical University of Warsaw, Warsaw, Poland *Author for correspondence: [email protected] 1 2
10.2217/FMB.14.126 © 2015 Future Medicine Ltd
Future Microbiol. (2015) 10(2), 199–204
part of
ISSN 1746-0913
199
Perspective Górski, Dąbrowska, Hodyra-Stefaniak et al. The correlation between the ability of the mutant phage to escape capture by the RES and the ability to rescue bacteremic mice suggests that such a phenomenon may improve the efficacy of phage therapy [16] . However, it is unclear whether this approach might also be useful in treating localized infections where vascular exit and parenchymal spread are necessary. In addition, it is unclear whether long persisting phagemia do not cause side effects (e.g., changes in immune reactivity and hematologic system, among others), which could adversely influence the final effect of therapy. The spread of phages throughout the organism following their intravenous administration (and using other routes) has been studied in some detail in mice and was summarized by Dabrowska et al. [18] . In particular, while significant titers of phages were present in the lung, kidney, spleen and liver, they were approximately 100-times lower than in the blood. In Matsuzaki’s experiments, following intraperitoneal (ip.) administration, phage can be detected in these organs in concentrations of 10–106 PFU/g [19] . When given orally to mice and human volunteers, even without an antacid, doses as low as 103 PFU/ml led to detection of phage in the feces, suggesting that phage can cross the stomach barrier [20] . What is more, phage concentration in a particular organ also depends on the absence or presence of susceptible bacteria. For example, phages may persist in infected fish spleen up to 5 days after ip. administration but only up to 24 h in the spleen of noninfected fish, which possibly reflects phage multiplication in situ [21] . The pharmacology of phage therapy has been analyzed and discussed by Levin and Bull [22] , and Payne and Janse [23,24] . Recently, Abedon has offered another in-depth analysis that introduced the term ‘inundation threshold’ to indicate the minimum phage density that can keep a bacterial infection from getting worse and, precisely, the density that keeps a bacterial culture from becoming more concentrated [25] . This is equivalent to the density that allows for bacterial killing in equilibrium with bacterial replication. According to the author’s calculations, the inundation threshold is equal to at least 107 phages/ml, but for a vigorously growing infection higher densities may be necessary. A more practical measure of phage ability to counteract infection is the killing titer (the density required to clear bacteria),
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which – according to Abedon – should be at least 108 phages/ml; achieving such phage densities over the course of treatment should be the dosing goal in phage therapy protocols [25] . Consequently, an important goal of optimal phage therapy protocol is to apply phages more directly to infections [25] . As stated, phage therapy efficiency is highly dependent on attaining a high phage ‘killing titer.’ If bacteria infiltrate to locations which phages are less able to penetrate, phage densities resulting from therapeutic administration may not be sufficient to achieve bacterial eradication. Thus, self amplification of phage in situ may be a mechanism enabling the therapeutic effect. However, it is unclear whether phage replication may always take place in infected organs at least to boost phage numbers to sufficient levels causing efficient elimination of bacteria. Therefore, as pointed out by Abedon, sufficiently high or repeated phage doses should be administered, rather than expecting that phage reproduce in situ, to achieve therapy success [25,26] . This effect should be achieved by a slow intravenous drip of a highly concentrated phage preparation. Notably, using of anion-exchange chromatography phages can be purified and concentrated to 1013 PFU/ml [27,28] . Selective targeting of a systematically administered drug would enhance the efficacy of treatment, an approach that could be of special significance for improving the success of antimicrobial treatment, including phage therapy. With this in mind, we envisage a novel approach to phage therapy based on bacteriophages presenting organ-specific peptides targeting them to infected organs, which should significantly enhance the efficacy of the therapy, reduce its potential side effects and protect against the neutralizing action of the immune system (e.g., production of phageinactivating antibodies and phagocytosis by neutrophils/monocytes among others). Phage can also induce opsonins that may restore their vulnerability to the RES with subsequent decay; moreover, possible toxicity of phage–antibody complexes should also be considered [29] . The renal glomerulus is particularly predisposed to the deposition of antigen–antibody complexes, which may cause both acute and chronic glomerular injury with serum sickness, acute and chronic glomerulonephritis [30] . Notably, virus–antibody complexes may mediate vasculitis of different tissues including skin, k idney, peripheral and CNS and liver [31] .
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Phages targeting infected tissues: novel approach to phage therapy As shown by the Benhar group, phages can be targeted to specific bacteria: targeted phages chemically conjugated with antibiotics may be nontoxic and less immunogenic as a result of drug conjugation [32–34] . However, this interesting approach, which offers selectivity of antibiotic action, does not solve the problem of antibiotic resistance. The fundamental studies of the Ruoslahti group have revealed that the endothelia of vessels of different organs are molecularly different (vascular zip codes). Those studies used in vivo phage display: a library of phage displaying random peptides is injected systematically into mice, followed by removal of target organs, amplification of the bound phage and subjecting the amplified pool to another round of selection, with positive selection at the target tissue. Initially, the group has identified homing peptides for brain and kidney [35] ; subsequent work has allowed for identification of other organ-specific peptides by the use of phage libraries [36] . Such phage nanoparticles armed with tissue homing peptides can ensure the advantage of topical application of drugs: high local concentration at the site of disease process and low systemic exposure (referred to as synaptic drug delivery) [37] . Parenchymal cells frequently express the same marker as endothelium; moreover, peptides enhancing nanoparticle transport from endothelium to extravascular tissue have been discovered and shown to facilitate nanoparticle penetration of tissues. Importantly, this system allowing phage extravasation and tissue penetration can be activated in a tissue-specific manner [37] . Use of such system could be very important in view of some experimental data suggesting inadequate capacity of unmodified phages to get out of the bloodstream and penetrate tissues [25] . Thus, the available data suggest that methods aiming at improved transport of phages to infected tissue may enhance the efficacy of phage therapy. At least three lines of evidence support this assumption. First, our knowledge about phage penetration to tissues following administration by routes other than topical is limited (almost nonexistent in man) and the available data are discordant. The prospects for acquiring such data in man in the near future look rather gloomy: lack of funding to carry out clinical trials and preclinical studies including pharmacokinetics in man constitute significant obstacles for progress in this area. The pharma industry is reluctant to fund such
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Perspective
studies in view of the lack of a specific frame for phage therapy under the current Medicinal Product Regulation. Also, in the United States, the amount of research and testing required by the US FDA is hampering the resurgence of phage therapy [38] . Second, the available data do not suggest that any route of administration could ascertain that tissue density of phages may reach the values suggested by Abedon to assure efficient local elimination of bacteria. Third, two ongoing clinical trials (one funded by the EC and the other by Nestle), as far as we are aware, do not include detailed pharmacokinetic studies [39,40] . On the other hand, recent decisions of the FDA facilitating access to experimental therapy ‘expanded access’ [41] and the increasing problem of microbial antibiotic resistance strongly suggest that phage therapy will continue to have a place in the treatment of patients in whom no other treatment is available and could find a much wider application – provided clinical trials confirm its efficacy. Many phages are polyvalent and infect members of several bacterial genera with phylogenetic relationship, which is relevant for phage therapy [42] . Both polyvalent with a broad host range and monovalent phages are used in therapy. At our center, it is possible to find an active specific phage for 90% of S. aureus, 72% of Pseudomonas aeruginosa and 77% of Enterococcus faecalis strains [14] . From the data presented above, it seems that phages presenting homing peptides (applied alone or perhaps along with transport-enhancing peptides) should allow for attaining higher local tissue concentrations of phages than those achievable using wild phages, and may therefore be an interesting alternative to currently used therapy, at least in infections where phage penetration could be reasonably expected to be poor (e.g., bone and prostate, among others). While there are no convincing data indicating that phage can cross the intestinal wall and migrate to blood or lymph in humans, a phenomenon known as bacterial translocation can also include phages [43] ; if this is indeed the case, oral administration of tissue-targeting phages might be a convenient and efficient novel form of phage therapy. The range of pathogens that can be eliminated by the targeted phages is directly related to the progress in phage engineering. Availability of molecular systems for phage modifications is the main limitation here. Currently, coliphages can be easily modified due to the abundance of Escherichia coli laboratory strains, coli-related vectors and
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Perspective Górski, Dąbrowska, Hodyra-Stefaniak et al. well established systems for phage display. Nevertheless, one may foresee upcoming solutions for constructing targeting phages against virtually any bacteria: synthetic genomics [44] will enable unlimited specificity of targeted phages. The idea of tissue-specific engineered phages is strongly supported by recent development of methodologies for phage engineering. ‘Classical’ phage display, based on introducing foreign gene sequences into phage genomes [45] , is not likely to offer a widely accepted therapeutic approach. This is because modified phages become genetically modified organisms. Environmental release of such constructs is strongly restricted and their use in general is not well accepted by the society. Some new types of phage display do not require introducing foreign DNA to phages. These can be in vitro modifications of mature phages as shown by Li et al. who reported the first presentation of anthrax toxin on bacteriophage T4 [46] . Other method is based on the competitive phage display: a phage propagates on a bacterial host that additionally to the phage produces also engineered fusions of phage proteins with active peptides or proteins. These fusions can be randomly incorporated into phage capsid, resulting in a modified phage particle without genetic manipulations in the phage [47,48] . Most currently used methods based on phage display use filamentous, lambda and T family phages, which ensure good peptide presentation while phage biological activity in infecting bacteria is maintained. In theory, other phages relevant for combating antibiotic-resistant infections could also be equipped with organ-specific homing receptors. However, the biological a ctivity of such constructs needs to be confirmed. Also chemical modifications, which do not employ phage display, can be used to anchor designed peptides on a phage surface. To date, this approach has been used mostly for nonprotein compounds [32,49] , but it could be further developed to modify phage surface with amino acidic motifs, for example, by dual conjugation of prosthetic groups. Our data indicating that phages can also mediate anti-inflammatory effects, which may be at least partly independent of their antibacterial action [50] , add another important dimension to this approach, as they suggest that organ-specific phages could mediate local anti-inflammatory action without significant side effects typical for currently used standard drugs (steroids and nonsteroidal anti-inflammatory agents). However,
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this promising effect needs to be validated by clinical trials. Conclusion In conclusion, the capacity of phages to leave the bloodstream, migrate to the infected tissues and achieve concentrations necessary to eradicate bacterial infection remains a significant challenge. This problem may be circumvented by constructing phages armed with tissue-specific homing peptides, which could be targeted to reach infected organs. This novel approach may significantly improve the effectiveness of phage therapy. Future perspective In the past decade, phage-expressing homing peptides have been shown to offer a novel approach to experimental therapy of a variety of disorders (cancer, atherosclerosis, tissue scarring). One could anticipate a similar pace of progress in the currently suggested field. Comparable time should allow for construction, experimental confirmation and perhaps initial clinical trials using phages targeted to penetrate bone and prostate, whose infection poses difficult medical challenges. The construction of phages specific for the most relevant pathogens and targeting organs and tissues could result in the establishment of a phage bank; the authors believe support for the costs of its operations should be sought from governmental or pharmaceutical company resources. Dedication This article is dedicated to the memory of Professor Ludwik Hirszfeld, founder of our Institute, who had contributed greatly to the development of phage therapy.
Financial & competing interests disclosure A Górski and B Weber-Dąbrowska are co-inventors on a patent covering phage preparations owned by the Institute. K Dąbrowska and A Górski are co-inventors on a patent application related to phage purification with the competitive phage display. This work was supported by National Science Centre in Poland (grant no. UMO-2014/13/N/ NZ6/03985) and by statutory funds of Medical University of Warsaw (1MG/N/2015). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
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Phages targeting infected tissues: novel approach to phage therapy
Perspective
EXECUTIVE SUMMARY ●●
Microbial antibiotic resistance constitutes one of the most challenging threats in medicine today. Given the slow rate of progress in the introduction of new drugs, phage therapy offers an interesting alternative to antibiotics; therefore, research and new developments in this area are urgently needed.
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The relatively short life of phages and their questionable tissue penetration warrant novel approaches enabling sufficient tissue density of phages to be attained to ensure effective eradication of infection.
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Phage display has been demonstrated to be an efficacious biotechnology tool yielding phages able to target specific
organs and tissues. Therefore, phage display could also be used to increase phage concentration in infected organs, which may further allow expansion of phage capability to reach specific pathogens that cause localized infections. This may be of special importance in case of tissues where penetration of antimicrobial agents is questionable. ●●
Regarding phage display, no data are available on the use of phages other than filamentous, lambda and T-family
phages; therefore, research is needed to confirm that such targeted phages also retain their efficient antibacterial activity. resistant bacteria. Adv. Exp. Med. Biol. 807, 97–110 (2014).
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