Pharmacokinetic and Pharmacodynamic Obstacles for Phage Therapy From the Perspective of Clinical Practice.

Bacteriophages present unique features that enable targeted killing of bacteria, including strains resistant to many antibiotics. However, phage pharmacokinetics and pharmacodynamics constitute much more complex and challenging aspects for researchers than those attributable to antibiotics. This is because phages are not just chemical substances, but also biological nanostructures built of different proteins and genetic material that replicate within their bacterial hosts and may induce immune responses acting as simple antigens. Here, we present a few examples of how primary general assumptions on phage pharmacokinetics and pharmacodynamics are verified by current preclinical and clinical observations, leading to conclusions that may not be obvious at first but are of significant value for the final success of phage therapy in humans.

Therapeutic Phages as Modulators of the Immune Response: Practical Implications.

While the medical community awaits formal proof of the efficacy of phage therapy, as is required by evidence-based medicine, existing data suggest that phages could also be applied based on their non-antibacterial action, especially phage-mediated immunomodulation. Promising avenues have been revealed by findings indicating that phages may mediate diverse actions in the immune system, while the list of phages able to dampen the aberrant immunity associated with a variety of disorders continuously grows. Here we summarize what is known in this field and possible options for the future. While available data are still scarce and preliminary, it appears that "phage repurposing" is worthy of more research, which could reveal new perspectives on applying phage therapy in contemporary medicine.

How Interest in Phages Has Bloomed into a Leading Medical Research Activity in Poland.

Poland has a leading position in phage therapy, as reflected by the number of patients treated and relevant publications in quality journals. The Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences was established by Ludwik Hirszfeld, a prominent microbiologist and serologist who also initiated studies on phages and pioneered the activities that set into motion phage therapy at the Institute. To achieve this goal, Hirszfeld had to overcome many difficulties in a post-war Poland. He died a month before the official start of the Institute's activity and was not able to witness the advancement of the Institute bearing his name. However, his hard work and dedication have been recently rewarded. In a recent evaluation of scientific performance, the Institute received the highest ranking in medical sciences among all universities and research institutions in Poland. One could consider it a posthumous tribute to the memory of L. Hirszfeld, being well-deserved on the grounds of the Institute's achievements (especially in the field of phage therapy) as well as his life and work.

Do Anti-Phage Antibodies Persist after Phage Therapy? A Preliminary Report.

Phages are immunogenic and may evoke an immune response following their administration. Consequently, patients undergoing phage therapy (PT) produce phage-neutralizing serum antibodies. The clinical significance of this phenomenon for the success or failure of the therapy is currently unclear. Interestingly, even a strong anti-phage humoral response does not exclude the success of PT. On the other hand, it cannot be ruled out that phage-antibody complexes may be trapped in tissues and organs causing injury and late complications of PT. Therefore, patients should be monitored for the presence of serum antibodies and therapy discontinued if their level is high. Our preliminary data suggest that the kinetics of the disappearance of those antibodies may vary from patient to patient and in some cases may take more than a year.

A Thorough Synthesis of Phage Therapy Unit Activity in Poland-Its History, Milestones and International Recognition.

The year 2020 marked 15 years of the Phage Therapy Unit in Poland, the inception of which took place just one year after Poland's accession to the European Union (2004). At first sight, it is hard to find any connection between these two events, but in fact joining the European Union entailed the need to adapt the regulatory provisions concerning experimental treatment in humans to those that were in force in the European Union. These changes were a solid foundation for the first phage therapy center in the European Union to start its activity. As the number of centers conducting phage therapy in Europe and in the world constantly and rapidly grows, we want to grasp the opportunity to take a closer look at the over 15-year operation of our site by analyzing its origins, legal aspects at the local and international levels and the impressive number and diversity of cases that have been investigated and treated during this time. This article is a continuation of our work published in 2020 summarizing a 100-year history of the development of phage research in Poland.

Bacteriophages and antibiotic interactions in clinical practice: what we have learned so far.

Bacteriophages (phages) may be used as an alternative to antibiotic therapy for combating infections caused by multidrug-resistant bacteria. In the last decades, there have been studies concerning the use of phages and antibiotics separately or in combination both in animal models as well as in humans. The phenomenon of phage-antibiotic synergy, in which antibiotics may induce the production of phages by bacterial hosts has been observed. The potential mechanisms of phage and antibiotic synergy was presented in this paper. Studies of a biofilm model showed that a combination of phages with antibiotics may increase removal of bacteria and sequential treatment, consisting of phage administration followed by an antibiotic, was most effective in eliminating biofilms. In vivo studies predominantly show the phenomenon of phage and antibiotic synergy. A few studies also describe antagonism or indifference between phages and antibiotics. Recent papers regarding the application of phages and antibiotics in patients with severe bacterial infections show the effectiveness of simultaneous treatment with both antimicrobials on the clinical outcome.

Phage therapy of wound-associated infections.

Phages are viruses which can specifically infect bacteria, resulting in their destruction. Bacterial infections are a common complication of wound healing, and experimental evidence from animal models demonstrates promising potential for phage-dependent eradication of wound-associated infections. The studies discussed suggest that phage therapy may be an effective treatment, with important advantages over some current antibacterial treatments. Phage cocktails, as well as co-administration of phages and antibiotics, have been reported to minimise bacterial resistance. Further, phage-antibiotic synergism has been reported in some studies. The ideal dose of phages is still subject to debate, with evidence for both high and low doses to yield therapeutic effects. Novel delivery methods, such as hydrogels, are being explored for their advantages in topical wound healing. There are more and more Good Manufacturing Practice facilities dedicated to manufacturing phage products and phage therapy units across the world, showing the changing perception of phages which is occurring. However, further research is needed to secure the place of phages in modern medicine, with some scientists calling upon the World Health Organisation to help promote phage therapy.

Bronislawa Fejgin (1883-1943): Forgotten Important Contributor to International Microbiology and Phage Therapy.

Bronislawa Brandla Fejgin was a Polish-born Jewish female physician. Among Fejgin's numerous articles in the field of microbiology, her later work was almost entirely devoted to phage research. Although not equally famous as the phage pioneers from Western Europe, F.W. Twort and F. d'Herelle, Fejgin's contribution to phage research deserves proper recognition. Her studies on phages resulted in the publication of numerous original scientific reports. These articles, published mostly in French, constitute an important source of information and expertise on early attempts towards therapeutic use of phages in humans. The interwar period marks the most intense years in Bronislawa Fejgin's research activity, brutally interrupted by her death in the Warsaw Ghetto in 1943. Her microbiology contributions have not been analyzed so far. Thus, the aim of this article is to fill the existing gap in the history of microbiology and phage therapy.

Potential for Phages in the Treatment of Bacterial Sexually Transmitted Infections.

Bacterial sexually transmitted infections (BSTIs) are becoming increasingly significant with the approach of a post-antibiotic era. While treatment options dwindle, the transmission of many notable BSTIs, including Neisseria gonorrhoeae, Chlamydia trachomatis, and Treponema pallidum, continues to increase. Bacteriophage therapy has been utilized in Poland, Russia and Georgia in the treatment of bacterial illnesses, but not in the treatment of bacterial sexually transmitted infections. With the ever-increasing likelihood of antibiotic resistance prevailing and the continuous transmission of BSTIs, alternative treatments must be explored. This paper discusses the potentiality and practicality of phage therapy to treat BSTIs, including Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Streptococcus agalactiae, Haemophilus ducreyi, Calymmatobacterium granulomatis, Mycoplasma genitalium, Ureaplasma parvum, Ureaplasma urealyticum, Shigella flexneri and Shigella sonnei. The challenges associated with the potential for phage in treatments vary for each bacterial sexually transmitted infection. Phage availability, bacterial structure and bacterial growth may impact the potential success of future phage treatments. Additional research is needed before BSTIs can be successfully clinically treated with phage therapy or phage-derived enzymes.

Immune Response to Therapeutic Staphylococcal Bacteriophages in Mammals: Kinetics of Induction, Immunogenic Structural Proteins, Natural and Induced Antibodies.

Bacteriophages are able to affect the human immune system. Phage-specific antibodies are considered as major factors shaping phage pharmacokinetics and bioavailability. So far, general knowledge of phage antigenicity nevertheless remains extremely limited. Here we present comparative studies of immunogenicity in two therapeutic bacteriophages, A3R and 676Z, active against Staphylococcus aureus, routinely applied in patients at the Phage Therapy Unit, Poland. Comparison of the overall ability of whole phages to induce specific antibodies in a murine model revealed typical kinetics of IgM and IgG induction by these two phages. In further studies we identified the location of four phage proteins in the virions, with the focus on the external capsid head (Mcp) or tail sheath (TmpH) or an unidentified precise location (ORF059 and ORF096), and we confirmed their role as structural proteins of these viruses. Next, we compared the immune response elicited by these proteins after phage administration in mice. Similar to that in T4 phage, Mcp was the major element of the capsid that induced specific antibodies. Studies of protein-specific sera revealed that antibodies specific to ORF096 were able to neutralize antibacterial activity of the phages. In humans (population level), none of the studied proteins plays a particular role in the induction of specific antibodies; thus none potentially affects in a particular way the effectiveness of A3R and 676Z. Also in patients subjected to phage therapy, we did not observe increased specific immune responses to the investigated proteins.

Low Immunogenicity of Intravesical Phage Therapy for Urogenitary Tract Infections.

Patients with chronic urinary and urogenital multidrug resistant bacterial infections received phage therapy (PT) using intravesical or intravesical and intravaginal phage administration. A single course of PT did not induce significant serum antibody responses against administered phage. Whilst the second cycle of PT caused a significant increase in antibody levels, they nevertheless remained quite low. These data combined with good therapy results achieved in some patients suggest that this mode of PT may be an efficient means of therapy for urogenital infections and a reliable model for a clinical trial of PT.

Treatment of recurrent urinary tract infections in a 60-year-old kidney transplant recipient. The use of phage therapy.

We would like to demonstrate the difficulty of treatment in a patient after kidney transplantation (KTX) who developed chronic urinary tract infection (UTI) with a multi-drug resistant ESBL-producing Klebsiella pneumoniae. The patient underwent several treatment interventions including supportive therapy with bacteriophages. This article presents a case of a 60-year-old patient after KTX repeatedly admitted to the hospital with recurrent UTIs caused by ESBL-producing Klebsiella pneumoniae showing variable susceptibility to carbapenems and full susceptibility to colistin only. KTX was performed due to renal insufficiency caused by polycystic kidney disease. The patient experienced 12 severe episodes of UTI due to K pneumoniae within 15 months since transplantation. In an attempt to curb the ongoing infections, phage therapy (PT) was applied on the experimental basis, coordinated by the Phage Therapy Unit of the Hirszfeld Institute in Wroclaw, Poland. Eventually, the patient fully recovered following nephrectomy of his own left kidney where cysts were the suspected reservoir of bacteria. The patient completed 29 days of PT. PT caused no reported side effects in the described case of the KTX recipient, although its role in controlling chronic UTI caused by K pneumoniae is unclear. More studies are needed in the population of kidney transplant recipients.

Therapeutic Perspectives and Mechanistic Insights of Phage Therapy in Allotransplantation.

Bacterio(phages) are bacteria-infecting viruses that employ host translation machinery to replicate, and upon cell lysis, release new particles into the environment. As a result, phages are prey-specific, thus making targeted phage therapy (PT) possible. Indeed, pre- and posttransplant bacterial infections pose a substantial risk to allograft recipients in their clinical course. Moreover, with the increasing threat of antibiotic resistance, the interest in PT as a potential solution to the crisis of multidrug-resistant bacterial pathogens has rapidly grown. Although little is known about the specific characteristics of the phage-directed immune responses, recent studies indicate phages exert anti-inflammatory and immunomodulatory functions, which could be beneficial in allotransplantation (allo-Tx). PT targeting multidrug-resistant Klebsiella pneumoniae, Mycobacterium abscessus, and Pseudomonas aeruginosa have been successfully applied in renal, lung, and liver allo-Tx patients. In parallel, the gastrointestinal microbiota appears to influence allo-Tx immunity by modulating the endoplasmic reticulum stress and autophagy signaling pathways through hepatic EP4/CHOP/LC3B platforms. This review highlights the current relevant immunobiology, clinical developments, and management of PT, and lays the foundation for future potential standard care use of PT in allo-Tx to mitigate early allograft dysfunction and improve outcomes. In conclusion, with novel immunobiology and metabolomics insights, harnessing the potential of PT to modulate microbiota composition/diversity may offer safe and effective refined therapeutic means to reduce risks of infections and immunosuppression in allo-Tx recipients.

Phage Prevalence in the Human Urinary Tract-Current Knowledge and Therapeutic Implications.

Recent metagenomic analyses imply an immense abundance of phages in the human body. Samples collected from different sites (lungs, skin, oral cavity, intestines, ascitic fluid, and urine) reveal a generally greater number of phage particles than that of eukaryotic viruses. The presence of phages in those tissues and fluids reflects the paths they must overcome in the human body, but may also relate to the health statuses of individuals. Besides shaping bacterial metabolism and community structure, the role of phages circulating in body fluids has not been fully understood yet. The lack of relevant reports is especially visible with regard to the human urobiome. Certainly, phage presence and the role they have to fulfill in the human urinary tract raises questions on potential therapeutic connotations. Urinary tract infections (UTIs) are among the most common bacterial infections in humans and their treatment poses a difficult therapeutic dilemma. Despite effective antibiotic therapy, these infections tend to recur. In this review, we summarized the recent data on phage presence in the human urinary tract and its possible implications for health and disease.

Phage Therapy: Towards a Successful Clinical Trial.

While phage therapy carried out as compassionate use (experimental therapy) has recently flourished, providing numerous case reports of supposedly healed patients, clinical trials aiming to formally prove their value in accord with current regulatory requirements have failed. In light of the current issue of increasing antibiotic resistance, the need for a final say regarding the place of phage therapy in modern medicine is evident. We analyze the possible factors that may favor success or lead to the failure of phage therapy: quality of phage preparations, their titer and dosage, as well as external factors that could also contribute to the outcome of phage therapy. Hopefully, better control of these factors may eventually bring about long-awaited positive results.

Sepsis, Phages, and COVID-19.

Phage therapy has emerged as a potential novel treatment of sepsis for which no decisive progress has been achieved thus far. Obviously, phages can help eradicate local bacterial infection and bacteremia that may occur in a syndrome. For example, phages may be helpful in correcting excessive inflammatory responses and aberrant immunity that occur in sepsis. Data from animal studies strongly suggest that phages may indeed be an efficient means of therapy for experimentally induced sepsis. In recent years, a number of reports have appeared describing the successful treatment of patients with sepsis. Moreover, novel data on the anti-viral potential of phages may be interpreted as suggesting that phages could be used as an adjunct therapy in severe COVID-19. Thus, clinical trials assessing the value of phage therapy in sepsis, including viral sepsis, are urgently needed.

Phage Therapy in Poland - a Centennial Journey to the First Ethically Approved Treatment Facility in Europe.

Although phage discovery is an unquestionable merit of the English bacteriologist Frederick W. Twort and the Canadian-French microbiologist Félix d'Hérelle, who both discovered phages over 100 years ago, the Polish history of phage studies also dates back to those years. In contrast to the Western world, developing phage treatment in Poland has never been abandoned despite the country's tense history marked by the Second World War (WWII) and the communism era. Today, Poland takes a prominent and remarkable place in the phage research area. Furthermore, established in 2005, the Phage Therapy Unit at the Hirszfeld Institute of Immunology and Experimental Therapy in Wroclaw, the first such center within European borders, has quickly become a model for other centers in the world facing the issue of widespread antibiotic resistance. This article constitutes an attempt to fill the gap in the scientific literature by providing a comprehensive summary of the long tradition of phage research in Poland.

Natural and Induced Antibodies Against Phages in Humans: Induction Kinetics and Immunogenicity for Structural Proteins of PB1-Related Phages.

Background: Bacteriophages may induce specific antibodies after natural exposure to phages or after phage therapy. As such, phage-specific antibodies might impact phage bioavailability in vivo, although limited non-neutralizing or insignificant effects have also been reported. Materials and Methods: Here, we report antibody induction against PB1-related phages (Pseudomonas viruses LMA2, F8, DP1) in mice over an 80-day period, for a healthy population of humans, and in patients undergoing phage therapy (oral and/or topical treatment). Results: All phages effectively induced specific immunoglobulin M and immunoglobulin G in mice. Phage-specific antibodies were observed in humans, whereas recombinant virion proteins (PB1 gp22, gp29) did not induce phage-neutralizing antibodies, either in mice or in humans. The healthy human population was differentiated for frequency of phage-neutralizing antibodies. Conclusions: These data can hold key considerations for phage therapy cocktail design, as highly similar phages can still be highly complementary in cases where specific immune response hinders therapeutic use of phages.