Phage therapy

thumb|Phage injecting its genome into bacterial cell

thumb|An [[electron micrograph of bacteriophages attached to a bacterial cell. These viruses are the size and shape of coliphage T1.]]

Phage therapy, viral phage therapy, or phagotherapy is the therapeutic use of bacteriophages for the treatment of pathogenic bacterial infections. that attach to bacterial cells and inject their genome into the cell. The bacteria's production of the viral genome interferes with its ability to function, halting the bacterial infection. Because phages replicate in vivo (in cells of living organism), a smaller effective dose can be used. Consequently, phage mixtures ("cocktails") are sometimes used to improve the chances of success. Ongoing challenges include the need to increase phage collections from reference phage banks, the development of efficient phage screening methods for the fast identification of the therapeutic phage(s), the establishment of efficient phage therapy strategies to tackle infectious biofilms, the validation of feasible phage production protocols that assure quality and safety of phage preparations, and the guarantee of stability of phage preparations during manufacturing, storage, and transport.

Phages tend to be more successful than antibiotics where there is a biofilm covered by a polysaccharide layer, which antibiotics typically cannot penetrate. However, the interactions between phages and biofilms can be complex, with phages developing symbiotic as well as predatory relationships with biofilms.

Phages are currently being used therapeutically to treat bacterial infections that do not respond to conventional antibiotics, particularly in Russia Phages are the subject of renewed clinical attention in Western countries, such as the United States. In 2019, the United States Food and Drug Administration approved the first US clinical trial for intravenous phage therapy.

Phage therapy has many potential applications in human medicine as well as dentistry, veterinary science, and agriculture. and by French microbiologist Felix d'Hérelle in 1917. A Georgian, George Eliava, was making similar discoveries. He travelled to the Pasteur Institute in Paris, where he met d'Hérelle, and in 1923, he founded the Institute of Bacteriology, which later became known as the George Eliava Institute, in Tbilisi, Georgia, devoted to the development of phage therapy. Phage therapy is used in Russia, Georgia and Poland, and was used prophylactically for a time in the Soviet army, most notably during the Second World War.

While knowledge was being accumulated regarding the biology of phages and how to use phage cocktails correctly, early uses of phage therapy were often unreliable. Soviet researchers continued to develop and to refine their treatments and to publish their research and results. However, due to the scientific barriers of the Cold War, this knowledge was not translated and did not proliferate across the world. The oldest use is, however, in human medicine. Phages have been used against diarrheal diseases caused by E. coli, Shigella, or Vibrio and against wound infections caused by facultative pathogens of the skin like staphylococci and streptococci. Recently, the phage therapy approach has been applied to systemic and even intracellular infections, and non-replicating phage and isolated phage enzymes like lysins have been added to the antimicrobial arsenal. However, actual proof for the efficacy of these phage approaches in the field or the hospital is not available. and in July 2015, the US National Institutes of Health hosted a two-day workshop titled "Bacteriophage Therapy: An Alternative Strategy to Combat Drug Resistance".

In January 2016, phages were used successfully at Yale University by Benjamin Chan to treat a chronic Pseudomonas aeruginosa infection in ophthalmologist Ali Asghar Khodadoust. This successful treatment of a life-threatening infection sparked a resurgence of interest in phage therapy in the United States.

In 2017, a pair of genetically engineered phages along with one naturally occurring (so-called "phage Muddy") each from among those catalogued by SEA-PHAGES (Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science) at the Howard Hughes Medical Institute by Graham Hatfull and colleagues, was used by microbiologist James Soothill at Great Ormond Street Hospital for Children in London to treat an antibiotic-resistant bacterial (Mycobacterium abscessus) infection in a young woman with cystic fibrosis.

In 2022, two mycobacteriophages were administered intravenously twice daily to a young man with treatment-refractory Mycobacterium abscessus pulmonary infection and severe cystic fibrosis lung disease. Airway cultures for M. abscessus became negative after approximately 100 days of combined phage and antibiotic treatment, and a variety of biomarkers confirmed the therapeutic response. The individual received a bilateral lung transplant after 379 days of treatment, and cultures from the explanted lung tissue confirmed eradication of the bacteria.

Potential benefits

thumb|Phage therapy is the use of bacteriophages to treat bacterial infections.

Bacteriophage treatment offers a possible alternative to conventional antibiotic treatments for bacterial infection. Viruses, just like bacteria, can evolve resistance to different treatments. The species and strain specificity of bacteriophages makes it unlikely that harmless or useful bacteria will be killed when fighting an infection.

A few research groups in the West are engineering a broader-spectrum phage and also a variety of forms of MRSA treatments, including impregnated wound dressings, preventative treatment for burn victims, and phage-impregnated sutures. Enzybiotics are a new development at Rockefeller University that create enzymes from phages. Purified recombinant phage enzymes can be used as separate antibacterial agents in their own right.

File:Bacteriophage plaquing host range values by bacteriophage families EN.svg|Proportions of bacterial strains supporting bacteriophage replication grouped by bacteriophage families

File:Bacteriophage plaquing host range values by bacterial species EN.svg|Proportions of bacterial strains supporting bacteriophage replication grouped by host species

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Applications

Collection

Phages for therapeutic use can be collected from environmental sources that likely contain high quantities of bacteria and bacteriophages, such as effluent outlets, sewage, or even soil.

Phage cocktails are commonly sold in pharmacies in Eastern European countries, such as Russia and Georgia.

Phages in practice are applied orally, topically on infected wounds or spread onto surfaces, or during surgical procedures. Injection is rarely used, avoiding any risks of trace chemical contaminants that may be present from the bacteria amplification stage, and recognizing that the immune system naturally fights against viruses introduced into the bloodstream or lymphatic system.

Reviews of phage therapy indicate that more clinical and microbiological research is needed to meet current standards.

In 2007, phase-1 and 2 clinical trials were completed at the Royal National Throat, Nose and Ear Hospital, London, for Pseudomonas aeruginosa infections (otitis).

Locus Biosciences has created a cocktail of three CRISPR-modified phages. A 2019 study examined its effectiveness against E. coli in the urinary tract, and a phase-1 trial was completed shortly before March 2021. In February 2019, the FDA approved the first clinical trial of intravenously administered phage therapy in the United States.

In July 2020, the FDA approved the first clinical trial of nebulized phage therapy in the United States. This double-blind, placebo-controlled study at Yale University will be focused on treating P. aeruginosa infections in patients with cystic fibrosis.

In February 2020, the FDA approved a clinical trial to evaluate bacteriophage therapy in patients with urinary tract infections. The study started in December 2020 and aims to identify ideal bacteriophage treatment regimens based on improvements in disease control rates.

In February 2021, the FDA approved a clinical trial to evaluate bacteriophage therapy in patients with chronic prosthetic joint infections (PJI). The study was to begin in October 2022 and be conducted by Adaptive Phage Therapeutics, in collaboration with the Mayo Clinic.

Administration

As pills

If administered as pills, phages can be freeze-dried; this procedure does not reduce efficacy. Bacteriophages are studied as potential candidates for treating bacterial lung infections, especially those caused by multidrug-resistant (MDR) bacteria. In these studies, bacteriophage solutions are administered via nebulizers, mostly using the compressor type. The stability and viability of phages during nebulization are crucial for their therapeutic efficacy. Current studies focus on whether phages can remain viable and effective when delivered via nebulizers. The choice of nebulizer can impact the stability and delivery efficiency of phages. Compressor nebulizers are commonly used because they generate a fine mist that can reach the lower respiratory tract.

In contrast to the compressor nebulizers, the ultrasound nebulizers can impact the viability of bacteriophages. The ultrasonic waves used to generate the aerosol can cause physical damage to the phages, potentially reducing their effectiveness. Preliminary research suggests the high-frequency vibrations and heat generated during the nebulization process can lead to a significant loss of phage activity. Consequently, one of the main challenges is ensuring that the phages remain undamaged during the nebulization process. Studies have shown that phages can be sensitive to the shear forces generated during nebulization. Still, with proper formulation and device selection, it is possible to maintain their viability, as the current research suggests.

Successful treatments

Phages were used successfully at Yale University by Benjamin Chan to treat a Pseudomonas infection in 2016. Nebulized phage therapy has been used successfully to treat numerous patients with cystic fibrosis and multidrug-resistant bacteria at Yale University as part of their compassionate use program. In 2019, a Brownsville, Minnesota resident with a longstanding bacterial infection in his knee received a phage treatment at the Mayo Clinic that eliminated the need for amputation of his lower leg. Individualised phage therapy was also successfully used by Robert T. Schooley and others to treat a case of multi-drug-resistant Acinetobacter baumannii in 2015. In 2022, an individually adjusted phage-antibiotic combination as an antimicrobial resistance treatment was demonstrated and described in detail. The scientists called for scaling up the research and for further development of this approach.

Treatment of biofilm infections

thumb|The different steps at which phages may disrupt biofilm formation. The biofilm surrounding the bacteria would inhibit the ability of antibiotics to reach bacteria, but may have less impact on the phages.

Phage therapy is being used to great effect in the treatment of biofilm infections, especially Pseudomonas aeruginosa and Staphylococcus aureus. From 78 recent cases of treatment of biofilm infections, 96% of patients saw clinical improvement using phage therapy, and 52% of patients saw complete symptom relief or a full expungement of the affecting bacteria. The emergence of BIMs has also been observed in vivo using different animal models, although this usually occurs later than in vitro (reviewed in). This fast adaptation of bacteria to phage attack is usually caused by mutations on genes encoding phage receptors, which include lipopolysaccharides (LPS), outer membrane proteins, capsules, flagella, and pili, among others. However, some studies suggest that when phage resistance is caused by mutations in phage receptors, this might result in fitness costs to the resistance bacterium, which will ultimately become less virulent. Moreover, it has been shown that the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Therefore, it is conceivable to think that phage therapy that uses phages that exert selection for multidrug-resistant bacteria to become antibiotic-sensitive could potentially reduce the incidence of antibiotic-resistant infections.

Besides the prevention of phage adsorption by loss or modification of bacterial receptors, phage insensitivity can be caused by: prevention of phage DNA entry by superinfection exclusion systems; or degradation of phage DNA by restriction-modification systems or by CRISPR-Cas systems; and use of abortive infection systems that block phage replication, transcription, or translation, usually in conjunction with suicide of the host cell. Altogether, these mechanisms promote a quick adaptation of bacteria to phage attack and therefore, the emergence of phage-resistant mutants is frequent and unavoidable.

It is still unclear whether the wide use of phages would cause resistance similar to what has been observed for antibiotics. In theory, this is not very likely to occur, since phages are very specific, and therefore, their selective pressure would affect a very narrow group of bacteria. However, we should also consider the fact that many phage resistance systems are mounted on mobile genetic elements, including prophages and plasmids, and thus may spread quite rapidly even without direct selection. Nevertheless, in contrast to antibiotics, phage preparations for therapeutic applications are expected to be developed in a personalized way because of the high specificity of phages. In addition, strategies have been proposed to counter the problem of phage resistance. One of the strategies is the use of phage cocktails with complementary host ranges (different host ranges, which, when combined, result in an overall broader host range) and targeting different bacterial receptors. Another strategy is the combination of phages with other antimicrobials such as antibiotics, disinfectants, or enzymes that could enhance their antibacterial activity. The genetic manipulation of phage genomes can also be a strategy to circumvent phage resistance.

Safety aspects

Bacteriophages are bacterial viruses, evolved to infect bacterial cells. To do that, phages must use characteristic structures at cell surfaces (receptors), and to propagate they need appropriate molecular tools inside the cells. Bacteria are prokaryotes, and their cells differ substantially from eukaryotes, including humans or animals. For this reason, phages meet the major safety requirement: they do not infect treated individuals. Even engineered phages and induced artificial internalization of phages into mammalian cells do not result in phage propagation. Natural transcytosis of unmodified phages, that is, uptake and internal transport to the other side of a cell, which was observed in human epithelial cells, did not result in phage propagation or cell damage. Recently, however, it was reported that filamentous temperate phages of P. aeruginosa can be endocytosed into human and murine leukocytes, resulting in transcription of the phage DNA. In turn, the product RNA triggers maladaptive innate viral pattern-recognition responses and thus inhibits the immune clearance of the bacteria. Whether this also applies to dsDNA phages like Caudovirales has not yet been established; this is an important question to be addressed as it may affect the overall safety of phage therapy.

Due to many experimental treatments in human patients conducted in past decades, and to already existing RCTs (see section: Clinical experience and randomized controlled trials), phage safety can be assessed directly. The first safety trial in healthy human volunteers for a phage was conducted by Bruttin and Brüssow in 2005. They investigated the oral administration of Escherichia coli phage T4 and found no adverse effects of the treatment.

Historical record shows that phages are safe, with mild side effects, if any. Macrophages, key cells of the innate immune system, play a central role in mediating this response. The most frequent (though still rare) adverse reactions to phage preparations found in patients were symptoms from the digestive tract, local reactions at the site of administration of a phage preparation, superinfections, and a rise in body temperature. These reactions might have occurred because either toxins were released from bacteria destroyed by the phages—such toxin release from bacteria can also happen with antibiotic use—or due to leftover bacterial fragments or residual components from the bacterial growth medium ("food for bacteria") present in the phage treatment when unpurified preparations were used.

When bacteriophages are introduced into the body, they may be recognized as foreign entities by macrophages through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). The binding of bacteriophages to these receptors triggers macrophage activation, leading to phagocytosis (macrophages engulf and digest the bacteriophages) and cytokine production: activated macrophages produce pro-inflammatory cytokines. These cytokines can modulate the immune response but generally do not result in significant fever when phages are used appropriately. Applying bacteriophages directly to the mucosa targets the site of infection with minimal systemic exposure, leading to a localized immune response. Injecting bacteriophages into muscle tissue introduces them to a larger number of macrophages in the muscle and regional lymph nodes. In intravenous injection, direct introduction into the bloodstream exposes bacteriophages to macrophages throughout the body, including those in the spleen and liver. However, significant elevations in body temperature are uncommon and typically only observed in cases of rapid phage administration or high doses. Macrophages are integral to the body's immune response to bacteriophage therapy, mediating any potential immune reactions. Intravenous administration of bacteriophages is conducted under strict medical supervision, by specialists in infectious diseases within a hospital setting, due to potential adverse reactions. Adverse reactions to intravenous bacteriophage therapy may include hypotension, i.e., a drop in blood pressure, leading to loss of consciousness. A sudden drop (chills) and rise (fever) in body temperature, known as the Jarisch–Herxheimer reaction, can occur due to the rapid lysis of bacteria and release of endotoxins. Rapid bacterial lysis releases endotoxins (e.g., lipopolysaccharides from gram-negative bacteria) that trigger systemic inflammatory responses, including "cytokine storms". Continuous monitoring of heart rate, blood pressure, and temperature to detect early signs of adverse reactions is done after the intravenous phage administration. Successful treatment of life-threatening infections with intravenous phage therapy has been documented. Patients have responded to therapy after one or several intravenous administrations, clearing infections that were unresponsive to conventional treatments: phages can disrupt biofilms, which are often resistant to antibiotics, enhancing infection clearance.

Bacteriophages must be produced in bacteria that are lysed (i.e., fragmented) during phage propagation. As such, phage lysates contain bacterial debris that may affect the human organism even when the phage itself is harmless. For these and other reasons, purification of bacteriophages is considered important, and phage preparations need to be assessed for their safety as a whole, particularly when phages are to be administered intravenously. This is consistent with general procedures for other drug candidates. In 2015, a group of phage therapy experts summarized the quality and safety requirements for sustainable phage therapy.

Phage effects on the human microbiome also contribute to safety issues in phage therapy. Many phages, especially temperate ones, carry genes that can affect the pathogenicity of the host. Even lambda, a temperate phage of the E. coli K-12 laboratory strain, carries two genes that provide potential virulence benefits to the lysogenic host, one that increases intestinal adherence and the other that confers resistance to complement killing in the blood. For this reason, temperate phages are generally to be avoided as candidates for phage therapy, although in some cases, the lack of lytic phage candidates and emergency conditions may make such considerations moot. Another potential problem is generalized transduction, a term for the ability of some phages to transfer bacterial DNA from one host to another. This occurs because the systems for packaging of the phage DNA into capsids can mistakenly package host DNA instead. Indeed, with some well-characterized phages, up to 5% of the virus particles contain only bacterial DNA. Thus in a typical lysate, the entire genome of the propagating host is present in more than a million copies in every milliliter. For these reasons, it is imperative that any phage to be considered for therapeutic usage should be subjected to thorough genomic analysis and tested for the capacity for generalized transduction.

As antibacterials, phages may also affect the composition of microbiomes, by infecting and killing phage-sensitive strains of bacteria. However, a major advantage of bacteriophages over antibiotics is the high specificity of bacteriophages. This specificity limits antibacterial activity to a sub-species level; typically, a phage kills only selected bacterial strains. For this reason, phages are much less likely (than antibiotics) to disturb the composition of a natural microbiome or to induce dysbiosis. This was demonstrated in experimental studies where microbiome composition was assessed by next-generation sequencing that revealed no important changes correlated with phage treatment in human treatments.

Much of the difficulty in obtaining regulatory approval is proving to be the risks of using a self-replicating entity that has the capability to evolve.

On their rediscovery, at the end of the 1990s, phage preparations were classified as medicines, i.e., "medicinal products" in the EU or "drugs" in the US. However, the pharmaceutical legislation that had been implemented since their disappearance from Western medicine was mainly designed to cater for industrially-made pharmaceuticals, devoid of any customization and intended for large-scale distribution, and it was not deemed necessary to provide phage-specific requirements or concessions.

Today's phage therapy products need to comply with the entire battery of medicinal product licensing requirements: manufacturing according to GMP, preclinical studies, phase I, II, and III clinical trials, and marketing authorisation. Technically, industrially produced predefined phage preparations could make it through the conventional pharmaceutical processes, minding some adaptations. However, phage specificity and resistance issues are likely to cause these defined preparations to have a relatively short useful lifespan. The pharmaceutical industry is currently not considering phage therapy products. Yet, a handful of small and medium-sized enterprises have shown interest, with the help of risk capital and/or public funding. Currently, no defined therapeutic phage product has made it to the EU or US markets.

thumb|Conventional drug development process vs. magistral preparation

According to Jean-Paul Pirnay, therapeutic phages should be prepared individually and kept in large phage banks, ready to be used, upon testing for effectiveness against the patient's bacterial pathogen(s). Intermediary or combined (industrially made as well as precision phage preparations) approaches could be appropriate.

Meanwhile, representatives from the medical, academic, and regulatory communities have established some (temporary) national solutions. For instance, phage applications have been performed in Europe under the umbrella of Article 37 (Unproven Interventions in Clinical Practice) of the Helsinki Declaration. To enable the application of phage therapy after Poland had joined the EU in 2004, the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy in Wrocław opened its own Phage Therapy Unit (PTU). Phage therapy performed at the PTU is considered an "experimental treatment", covered by the adapted Act of 5 December 1996 on the Medical Profession (Polish Law Gazette, 2011, No. 277 item 1634) and Article 37 of the Helsinki Declaration. Similarly, in the last few years, a number of phage therapy interventions have been performed in the US under the FDA's emergency Investigational New Drug (eIND) protocol.

Some patients have been treated with phages under the umbrella of "compassionate use", which is a treatment option that allows a physician to use a not-yet-authorized medicine in desperate cases. Under strict conditions, medicines under development can be made available for use in patients for whom no satisfactory authorized therapies are available and who cannot participate in clinical trials. In principle, this approach can only be applied to products for which earlier study results have demonstrated efficacy and safety, but have not yet been approved. Much like Article 37 of the Helsinki Declaration, the compassionate use treatment option can only be applied when the phages are expected to help in life-threatening or chronic and/or seriously debilitating diseases that are not treatable with formally approved products.

In France, ANSM, the French medicine agency, has organized a specific committee—Comité Scientifique Spécialisé Temporaire (CSST)—for phage therapy, which consists of experts in various fields. Their task is to evaluate and guide each phage therapy request that ends up at the ANSM. Phage therapy requests are discussed together with the treating physicians and consensus advice is sent to the ANSM], which then decides whether or not to grant permission. Between 2006 and 2018, fifteen patients were treated in France (eleven recovered) using this pathway.

In Belgium, in 2016 and in response to a number of parliamentary questions, Maggie De Block, the Minister of Social Affairs and Health, acknowledged that it is indeed not evident to treat phages as industrially made drugs, and therefore she proposed to investigate if the magistral preparation pathway could offer a solution.

The first phage therapy case in China can be traced back to 1958, at Shanghai Jiao Tong University School of Medicine. However, many regulations were not yet established back then, and phage therapy soon lost people's interest due to the prevalence of antibiotics, which eventually led to the antimicrobial resistance crisis. This prompted researchers in China as well as the Chinese government to pay attention to phage therapy again, and following the first investigator-initiated trial (IIT) by the Shanghai Institute of Phage in 2019, phage therapy rapidly flourished. Currently, commercial phage therapy applications must go through either one of two pathways. The first is for fixed-ingredient phage products. The second pathway is for personalized phage products, which need to go through IITs. This way, the products are considered restrictive medical technologies.

Application in other species

Animals

Phage therapy has been a relevant mode of treatment in animals for decades. It has been proposed as a method of treating bacterial infections in the veterinary medical field in response to the rampant use of antibiotics. Studies have investigated the application of phage therapy in livestock species as well as companion animals. Brigham Young University has been researching the use of phage therapy to treat American foulbrood in honeybees. Phage therapy is also being investigated for potential applications in aquaculture.

Plants

Phage therapy has been studied for bacterial spot of stonefruit, caused by Xanthomonas pruni (syn. X. campestris pv. pruni, syn. X. arboricola pv. pruni) in prunus species.

Steffanie Strathdee's book The Perfect Predator: An Epidemiologist's Journey to Save Her Husband from a Deadly Superbug, co-written with her husband, Thomas Patterson, was published by Hachette Book Group in 2019. It describes Strathdee's ultimately successful attempt to introduce phage therapy as a life-saving treatment for her husband, critically ill with a completely antibiotic-resistant Acinetobacter baumannii infection following severe pancreatitis.

References

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External links

iBiology video: Phage Therapy (2016)
Popular Science – "The Next Phage" (2009)