Essential Topics for the Regulatory Consideration of Phages as Clinically Valuable Therapeutic Agents: A Perspective from Spain
1. General Aspects of the Use of Phages as Antimicrobials
- Activity against antibiotic-resistant bacteria. Phages can infect and kill bacteria, including MDR strains . This is the most obvious advantage towards recovering phage therapy to fight antimicrobial resistance today. Moreover, the composition of the phages for therapy may be designed to impose an evolutionary trade-off in which the evolution of bacterial resistance to phage results in increased susceptibility to antibiotics . Under this rationale, the combination therapy of phages plus antibiotics has a remarkable potential to smartly tackle antibiotic resistance by both eliminating resistant strains and preventing the dissemination of resistance genes [7,8]. Many works have been published thus far where phage–antibiotic synergy, known as PAS effect, is reported. This suggests that combined therapy can be the safest and a more advantageous approach, as it minimizes resistance and virulence [9,10,11].
- Specificity. The high specificity of many phages towards their host bacterial strains makes them a highly selective therapy that prevents the dysbiosis of the healthy microbiota. Contrary to antibiotics, only strains of the same genus or species—and often just one or very few strains within a species—are susceptible to infection by a given phage, protecting the normal microbiota and reducing side effects . The specificity of phages lies in the bacterial receptor that the virions recognize through one or more receptor-binding proteins , and can be further driven by post-entry anti-phage defense mechanisms . Phages can be polyvalent (i.e., with a broad host range) if they recognize a receptor present in several bacteria or, alternatively, their specificity can be very restricted if they bind receptors exclusive to a single bacterial type. Another possibility is that the phage uses a receptor that is only expressed under certain conditions which therefore restricts its infectivity (e.g., the receptor of phage lambda is the maltose receptor, which is only expressed in the presence of maltose ).
- Multiplication at the site of the infection (auto-dosage). Phages can multiply at the site of the infection. Once the phages reach the targeted bacteria, they will replicate and generate progeny. Therefore, if sufficient phage particles are able to reach the infection site, phage therapy can be considered as auto-dosage treatment. Furthermore, once the infection is successfully controlled, phages would be eliminated in the absence of bacterial hosts. Thus, whenever an auto-dosed, “active” treatment is achieved, it can elicit the infection eradication by only a single administration .
- Ubiquity and diversity. Phages can be found in virtually any environment , and they play an utmost important role in ecosystems by regulating bacterial populations , including the human microbiota. The main practical consequence of such ubiquity and the concomitant diversity is the ease of discovery of novel phages, which contrasts with the currently slow antibiotic discovery rate.
- Evolvability. Phages are evolving entities and, therefore, can be optimized using directed evolution techniques. This opens up many possibilities compared to conventional treatments, which are stable chemical compounds. Phage evolvability can be exploited in many ways, such as increasing lytic capability, improving particle stability, expanding the host range, or counteracting bacterial resistance. For instance, the Appelmans’ protocol uses spontaneous mutation and recombination among phages present in a cocktail to produce phage variants capable of infecting initially non-susceptible bacterial strains [19,20].
- Safety. Humans are carriers of many different phages forming the phageome [21,22]. Their biological functions, beyond regulating bacterial populations, are not yet entirely clear . However, their widespread presence in the human body seems to be a good safety indicator. Moreover, there is evidence of phage safety from clinical trials and intake of phage-treated foods . A potential concern of phage therapy could be the release of bacterial endotoxins after lysis of the targeted bacterial cells. It should be noted, however, that similar observations have been made regarding conventional therapy with certain antibiotics , and that the current literature does not support detrimental inflammatory reactions upon phage administration. Phages may also enter tissues that are not the specific target of the treatment, but these interactions also do not appear to produce side effects .
- Phage-resistance. In the same way that resistance to antibiotics emerges, bacteria can become resistant to phage infection. The most common solution to address this involves the use of cocktails of different phages, rather than a single phage, and/or the “à la carte” selection of phages for each particular infectious isolate. This makes it much less likely that the host will become resistant to all phages at the same time . The so-called “step-by-step” technique is an interesting approach in which phages are isolated against phage-resistant bacterial mutants in successive screening steps to obtain other phages capable of infecting resistant variants. By this method, the natural antagonistic co-evolution that would occur upon treatment is mimicked prior to therapy, thus generating a phage cocktail able to infect both the original bacterium and the foreseeable resistant variants . Moreover, the emergence of phage-resistant bacteria is not always a disadvantage, since it sometimes involves a decrease of the fitness or virulence of the bacterial host , or may resensitize bacteria to antibiotics .
- Specificity. This feature can be a double-edged sword. Phage specificity requires careful susceptibility testing of each bacterial pathogen before treatment, which may be viewed as an issue for certain acute infections that require urgent action. In addition, this specificity may require either the development of large phage libraries and/or extensive sampling and screening efforts to provide sufficient coverage of bacterial diversity. This can be a daunting task and has posed major regulatory issues, since, according to the current framework, each individual phage should undergo review and approval. In addition, the eventual need to develop a different phage preparation for each bacterial pathogen, as a personalized medicine, reduces business profitability and can be viewed as a serious drawback by pharmaceutical companies. Again, phage cocktails targeting different receptors or different bacterial strains would be a potential solution.
- In vivo phage activity. There is not necessarily a correlation between the in vitro and in vivo behavior of a phage, particularly regarding its propagation ability. This is due both to the complexity of body fluids and the ecological in vivo interactions [29,30]. In addition, the phage propagation is dependent on the physiological state of the bacterial host, which may not be optimal for infection in vivo (for example, depending on whether the bacterium is embedded or not within a biofilm, the expression of receptor molecules, etc.) . Moreover, phages are bigger than antibiotics and, therefore, diffuse less efficiently. This limitation is aggravated in vivo, where multiple physical barriers are encountered. Therefore, the probability of infection at low phage and bacterial densities is low, and the threshold densities required to ensure phage infection may often require the administration of very high phage doses [32,33].
- Immune response. Since phages are made of biomacromolecules, they are potentially capable of eliciting an immune reaction upon administration . Generally, the immune reactions against phage components are not considered problematic for the individual under treatment, although they do have a relevant contribution to the outcome of phage therapy . On one hand, the immune response potentially causes the removal of phages from the system , although this effect may be overcome by adjusting such parameters as dosing, administration route, etc. On the other hand, synergism with the immune antibacterial response seems relevant for therapeutic success , although some evidence suggests that phage therapy can also be successfully applied in immunocompromised patients . To sum up, the interaction of phages with the immune system is complex and not yet well understood, although many unknown implications seem to affect the therapeutic efficacy without contradicting the presumed safety of phage therapy.
- Gene transfer. Phages potentially have the ability to modify the genome of the host bacteria, which may increase their virulence or dissemination of antibiotic resistance genes . Indeed, phages can mobilize large fragments of bacterial genomes at relatively high frequencies [40,41]. To date, it is not known whether the mobilized DNA is randomly selected or whether the transfer of some particular genes is favored, e.g., those related to virulence, survival, or fitness of the host strain. A relevant mechanism for phage-mediated transfer of particular genes is associated with lysogeny (the integration of the phage genetic material into the bacterial chromosome) . Therefore, this issue could be minimized by selecting exclusively virulent phages, as well as by analyzing phage genomes in detail to ensure that they do not contain genes encoding toxins or any other undesirable genes.
2. Obtaining Therapeutic Phage Preparations
2.1. Selection of Screening Host Strains and Phages Intended for Therapy
2.2. Small- and Large-Scale Production Processes
2.3. Purification of Phage Solutions
2.5. Formulation and Administration
- Oral administration is appropriate for gastrointestinal infectious diseases. In some cases, oral phages given without additional protection, as water-based liquid suspensions, reportedly survived gastric passage and were recovered in the feces [60,61,62]. The formulation efforts for liquid phage suspensions are typically minimal since phages are just prepared in sterile buffers such as phosphate-buffered saline (PBS), the bacterial growth medium, standard saline, or water [61,63,64,65]. More elaborate formulations specifically intended for oral administration can improve phage survival through the extreme conditions of the gastrointestinal tract. For example, encapsulation protects phages from the highly acidic stomach environment and digestive enzymes . Furthermore, their release can be triggered in a controlled manner, for example, pH-dependent release, with capsules programmed to become permeable at different pHs regarding the aimed site of action: from the stomach (pH 1–3) to the small intestine (pH 5.5–6.5) or the colon (pH 6.5–7.2) . A wide range of natural and synthetic polymers are available that offer considerable plasticity for tailoring phage encapsulation and subsequent release to different biomedical applications, including polysaccharides, natural or synthetic plastic polymers, liposomes, and micelles [68,69].
- Topical administration of phages is chosen for skin infections, wounds, burns, ulcers, and osteoarticular infections . Phages have been topically administered in liquid, semi-solid, and liposome-encapsulated formulations, as well as phage-immobilized wound dressings . When using liquid preparations, they may just be dripped onto the infected site or applied in a gauze soaked with the preparation. Alternatively, gel or cream formulations are suitable to overcome some of the limitations of liquid preparations, with a preference for hydrogels over organic solvent-based gels. This is especially relevant for the treatment of burn wounds, since hydrogels help keep the wound hydrated as much as they favor phage stability . Commercial infection-care products can also be used as a formulation basis for topically delivering phages, but care should be taken as to whether the composition of the product reduces phage infectivity .
- The local phage treatment of respiratory infections requires preparing phages either as stable liquid formulations for intranasal instillation or nebulization, or as a solid powder in an inhalable form . The most popular formulations for respiratory infections are liquid suspensions, due to the relative simplicity of preparation. Nebulization of liquid phage suspensions has been tested with mixed outcomes, generally suggesting that temperature, relative humidity, the nebulization-induced mechanical stress, delivery efficiency of the system, and the nature of the phage itself greatly influence the outcome. Regarding dry powder inhalation, the methods to obtain solid phage formulations include freeze-drying or spray-drying. In general, both processes subject phages to diverse stresses that may impact their infectivity , but the control of key parameters and addition of suitable excipients, including polymers for encapsulation, can enhance phage preservation [74,75].
3. Quality Criteria for Therapeutic Phage Preparations
- Phage identity. The identity of each phage is defined by its specific genomic sequence [79,80]. Metagenomics has already been proposed as a quality control method for some vaccines , and thus has also been used to assess the composition of commercial phage products [82,83]. This method allows the detection of biological contaminants while also assuring the active product identity. While random mutations during propagation are inevitable, they need to be as limited as possible by process design (e.g., minimizing subcultivation steps), and functional properties should be regularly tested with validated quality controls, as even single-nucleotide polymorphisms can lead to significant phenotypic changes. However, a highly discriminating PCR-based genotyping technique might be sufficient in some cases . The maximum acceptable level of genomic divergence between the master batch and the phage population in the therapeutic product, as well as the frequency of such quality check, should be nonetheless adjusted on a case-by-case basis .
- Phage Titer. The titer of each individual phage is classically assessed by the double-layer agar method. An alternative is lethality curves, in which the kinetics of phage-induced lysis are assessed by measuring the optical density of phage-infected bacterial cultures . Other methods, such as qPCR and ELISA, can be used to quantify phages, but they do not necessarily quantify infectious viral particles, whereas double-layer and lethality assays do determine biological activity .
- General Purity. For biopharmaceuticals, the purity and correct composition is classically assessed by high-performance liquid chromatography, combined with mass spectrometry if necessary. These methods can be used to identify phage capsid proteins, toxins, or other bacterial proteins. Because of the potential risk posed by the necessary production with pathogenic bacterial hosts, quality criteria should specify maximum levels for contaminants such as toxins or bacterial DNA, which normally must be tested with specific and appropriate molecular biology methods as specified below.
- Toxins. Several in vitro methods have been developed for endotoxins quantification: gel-clot, turbidimetric, and chromogenic tests. Among the latter, the limulus amebocyte lysate assay is the most widely used . When this assay is not applicable, e.g., due to masking effect, a reporter cell line can be used . In addition, several commercial assays can be used to detect other toxic bacterial proteins, including ELISA or assays based on reporter cell lines.
- Contaminating Nucleic Acids. Quality controls may also be required to determine the concentration of contaminating nucleic acids (i.e., non-phage nucleic acids). The presence and concentration of residual nucleic acids are typically checked by qPCR.
- Other Quality Controls. Current regulations on sterility or general quality parameters in pharmaceutical products should also apply to phage-based pharmaceuticals . Some parameters that may need to be checked are the total microbial load, pH, osmolarity, visual appearance, and/or maximum water content (in lyophilized preparations) .
4. Regulation for Phage Preparations
5. Clinical Trials and Prospects for Phage Therapy
6. Most Urgent Indications for the Application of Phage Therapy in Spain
- In the case of a severe infection produced by an MDR bacterium.
- When the infection occurs in an area reluctant to the use of antibiotics, such as in prosthetics.
- Or, in general, whenever there is no standard of care option available, such as patients suffering from hypersensitivity to the antibiotic treatment.
6.1. Cystic Fibrosis
6.2. Osteoarticular Infections
7. General Aspects of the Use of Endolysins as Antimicrobials
8. Global Phage Therapy Market
9. Final Conclusions
Conflicts of Interest
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|Diabetic foot ulcers||Staphylococcus aureus||Topical phage cocktail||Not yet recruiting (expected start date: June 2022)||NCT02664740|
|Invasive infection in patients with inactive Crohn’s disease||E. coli||Oral phage cocktail||Recruiting (estimated completion: June 2023)||NCT03808103|
|Chronic airway infection in cystic fibrosis patients||P. aeruginosa||Nebulized phage therapy||Recruiting (estimated completion: December 2022)||NCT04684641|
|Diabetic foot ulcers||P. aeruginosa, S. aureus and/or Acinetobacter baumannii||Topical phage cocktail||Recruiting (estimated completion: December 2021||NCT04803708|
|Prosthetic joint infections||Several pathogens||Combined antibiotic/personalized phage therapy||Not yet recruiting (estimated start date: October 2022)||NCT04787250|
|Chronic airway infection in cystic fibrosis patients||P. aeruginosa||Nebulized phage cocktail||Not yet recruiting||NCT05010577|
|Wound infections in burned patients||S. aureus, P. aeruginosa or Klebsiella pneumoniae||Topical phage cocktail||Not yet recruiting (estimated start date: January 2022)||NCT04323475|
|Pressure injury infections||S. aureus, P. aeruginosa, K. pneumoniae||Topical phage cocktail in combination with antibiotics||Not yet recruiting (estimated start date: January 2022)||NCT04815798|
|Urinary tract infections||E. coli or K. pneumoniae||Personalized phage therapy administered through intravenous or intravesical route||Recruiting (estimated completion: September 2023)||NCT04287478|
|Tonsillitis||Several pathogens||Nebulized phage cocktail||Phase 3. Active, not recruiting (estimated completion: December 2024)||NCT04682964|
|Chronic airway infection in cystic fibrosis patients||P. aeruginosa||Inhaled phage cocktail||Recruiting (estimated completion: March 2022)||NCT04596319|
|CF with chronic MDR lung infection||Achromobacter|
|Inhalation, orally||Dyspnea resolved and cough reduced. Lung function improved|||
|CF with disseminated infection, lung transplantation||M. abscessus||Intravenous||Sternal wound closure, improved liver function, substantial resolution of infected skin nodules|||
|CF with MDR pneumonia, persistent respiratory failure, and colistin-induced renal failure||P. aeruginosa||Intravenous||Pneumonia clinically resolved, no sputum production, return to baseline renal function, white blood cell count normalized|||
|CF with persistent lung infection, lung transplantation||A. xylosoxidans||Inhalation||Respiratory condition improved; sputum cultures positive but with low bacteria concentration|||
|Lung transplant recipient patients with MRD resistant infections||P. aeruginosa and Burkholderia dolosa||Intravenous, inhalation||Two patients were discharged from the hospital off ventilator support. A third patient infection relapsed and died|||
|COPD with drug-resistant pneumonia||A. baumannii||Inhalation||Sputum/ blood and bronchoalveolar lavage fluid negative, restoration sinus rhythm, lung function improved|||
|Prosthesis infection||S. aureus||Local||Bacteria removed, rapid healing|||
|Osteomyelitis||P. aeruginosa||Local||No clinical signs of persistent infection|||
|Infection of the right knee and chronic osteomyelitis of the femur after injury||P. aeruginosa||Local||No pain, soft tissue at the surgical site unremarkable, mobility satisfactory|||
|Osteomyelitis of the distal phalanx||S. aureus||Local||The ulcer healed, re-ossification of the distal phalanx, erythema and edema decreased|||
|Fracture-related infection||K. pneumoniae||Local||Skin graft vascularized and viable, the sinus tract closed and dry, pus no longer discharged from the pin sites of the external fixator, restored muscle function|||
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Vázquez, R.; Díez-Martínez, R.; Domingo-Calap, P.; García, P.; Gutiérrez, D.; Muniesa, M.; Ruiz-Ruigómez, M.; Sanjuán, R.; Tomás, M.; Tormo-Mas, M.Á.; García, P. Essential Topics for the Regulatory Consideration of Phages as Clinically Valuable Therapeutic Agents: A Perspective from Spain. Microorganisms 2022, 10, 717. https://doi.org/10.3390/microorganisms10040717
Vázquez R, Díez-Martínez R, Domingo-Calap P, García P, Gutiérrez D, Muniesa M, Ruiz-Ruigómez M, Sanjuán R, Tomás M, Tormo-Mas MÁ, García P. Essential Topics for the Regulatory Consideration of Phages as Clinically Valuable Therapeutic Agents: A Perspective from Spain. Microorganisms. 2022; 10(4):717. https://doi.org/10.3390/microorganisms10040717Chicago/Turabian Style
Vázquez, Roberto, Roberto Díez-Martínez, Pilar Domingo-Calap, Pedro García, Diana Gutiérrez, Maite Muniesa, María Ruiz-Ruigómez, Rafael Sanjuán, María Tomás, María Ángeles Tormo-Mas, and Pilar García. 2022. "Essential Topics for the Regulatory Consideration of Phages as Clinically Valuable Therapeutic Agents: A Perspective from Spain" Microorganisms 10, no. 4: 717. https://doi.org/10.3390/microorganisms10040717