Ewa Jończyk-Matysiak

Mammalian Host-Versus-Phage immune response determines phage fate in vivo

Emerging bacterial antibiotic resistance draws attention to bacteriophages as a therapeutic alternative to treat bacterial infection. Examples of phage that combat bacteria abound. However, despite careful testing of antibacterial activity in vitro, failures nevertheless commonly occur. We investigated immunological response of phage antibacterial potency in vivo. Anti-phage activity of phagocytes, antibodies, and serum complement were identified by direct testing and by high-resolution fluorescent microscopy. We accommodated the experimental data into a mathematical model. We propose a universal schema of innate and adaptive immunity impact on phage pharmacokinetics, based on the results of our numerical simulations. We found that the mammalian-host response to infecting bacteria causes the concomitant removal of phage from the system. We propose the notion that this effect as an indirect pathway of phage inhibition by bacteria with significant relevance for the clinical outcome of phage therapy.

Phage Therapy: Combating Infections with Potential for Evolving from Merely a Treatment for Complications to Targeting Diseases.

Antimicrobial resistance is considered to be one of the greatest challenges of medicine and our civilization. Lack of progress in developing new anti-bacterial agents has greatly revived interest in using phage therapy to combat antibiotic-resistant infections. Although a number of clinical trials are underway and more are planned, the realistic perspective of registration of phage preparations and their entering the health market and significantly contributing to the current antimicrobial crisis is rather remote. Therefore, in addition to planning further clinical trials, our present approach of phage treatment carried out as experimental therapy (compassionate use) should be expanded to address the growing and urgent needs of increasing cohorts of patients for whom no alternative treatment is currently available. During the past 11 years of our phage therapy center’s operation, we have obtained relevant clinical and laboratory data which not only confirm the safety of the therapy but also provide important information shedding more light on many aspects of the therapy, contributing to its optimization and allowing for construction of the most appropriate clinical trials. New data on phage biology and interactions with the immune system suggest that in the future phage therapy may evolve from dealing with complications to targeting diseases. However, further studies are necessary to confirm this promising trend.

Bacteriophage Procurement for Therapeutic Purposes

Bacteriophages (phages), discovered 100 years ago, are able to infect and destroy only bacterial cells. In the current crisis of antibiotic efficacy, phage therapy is considered as a supplementary or even alternative therapeutic approach. Evolution of multidrug-resistant and pandrug-resistant bacterial strains poses a real threat, so it is extremely important to have the possibility to isolate new phages for therapeutic purposes. Our phage laboratory and therapy center has extensive experience with phage isolation, characterization, and therapeutic application. In this article we present current progress in bacteriophages isolation and use for therapeutic purposes, our experience in this field and its practical implications for phage therapy. We attempt to summarize the state of the art: properties of phages, the methods for their isolation, criteria of phage selection for therapeutic purposes and limitations of their use. Perspectives for the use of genetically engineered phages to specifically target bacterial virulence-associated genes are also briefly presented.

Phages and immunomodulation

In the past years, the microbiome and its role in the pathophysiology of diseases have gained great interest. The progress of our knowledge in this field opens completely novel prospects for treating disorders, including those which are most challenging to medicine today. Of special interest are studies on the interactions of the microbiome with the immune system. Only recently has the presence of bacteriophages in the microbiome been highlighted, and their potential role in maintaining normal immunity has gained increasing attention. We summarize the available data pointing to the potential impact of phages in maintaining immunological homeostasis.

Antiphage activity of sera during phage therapy in relation to its outcome.

AIMnThe aim was to study the association between the phage neutralization of patients sera and the clinical outcome of phage therapy (PT).nnnPATIENTSnAbout 62 patients with various bacterial infections receiving PT as well as 30 healthy volunteers were studied.nnnMATERIALS & METHODSnAntiphage activity of sera (AAS) was examined using the phage neutralization test of different types of phages before and during PT in relation to the route of phage administration and correlated with the results of PT.nnnRESULTS & CONCLUSIONnThe analysis of the association between AAS level and clinical results indicated that the level of AAS is not correlated with the outcome of PT.

The Effect of Bacteriophage Preparations on Intracellular Killing of Bacteria by Phagocytes

Intracellular killing of bacteria is one of the fundamental mechanisms against invading pathogens. Impaired intracellular killing of bacteria by phagocytes may be the reason of chronic infections and may be caused by antibiotics or substances that can be produced by some bacteria. Therefore, it was of great practical importance to examine whether phage preparations may influence the process of phagocyte intracellular killing of bacteria. It may be important especially in the case of patients qualified for experimental phage therapy (approximately half of the patients with chronic bacterial infections have their immunity impaired). Our analysis included 51 patients with chronic Gram-negative and Gram-positive bacterial infections treated with phage preparations at the Phage Therapy Unit in Wroclaw. The aim of the study was to investigate the effect of experimental phage therapy on intracellular killing of bacteria by patients peripheral blood monocytes and polymorphonuclear neutrophils. We observed that phage therapy does not reduce patients phagocytes ability to kill bacteria, and it does not affect the activity of phagocytes in patients with initially reduced ability to kill bacteria intracellularly. Our results suggest that experimental phage therapy has no significant adverse effects on the bactericidal properties of phagocytes, which confirms the safety of the therapy.

Bacteriophages in the gastrointestinal tract and their implications

The gut microbiota plays an essential role in health and disease of humans. Bacteriophages are the most abundant members of the gut virobiota and display great diversity. Phages can translocate through the mucosa to lymph and internal organs and play a role as regulators of the bacterial population in the gut. Increasing abundance of phages in the gut mucosa may reduce colonization by bacteria. Moreover, phages may have an immunomodulatory role in the immune response in the human gut. The role of phages in inflammatory bowel disease (IBD) remains unknown. Phages may take part in the development of IBD, but there are also data suggesting the protective role of phages in the gut of patients with IBD. Furthermore, recent data suggest that phages may mediate the beneficial effects of fecal microbiota transplantation (FMT). Therefore, evidence is accumulating to highlight the protective immunomodulating activity of the gut phages.

In Vivo Studies on the Influence of Bacteriophage Preparations on the Autoimmune Inflammatory Process

Phage preparations used for phage therapy may have not only direct antibacterial action but also immunomodulating effects mediated by phages themselves as well as by bacterial antigens. Therefore phage application in patients with immune disorders, and especially with autoimmune diseases, requires special attention. The aim of this study was to investigate the effect of phage lysates (staphylococcal phages A3/R, phi200, and MS-1 cocktail, enterococcal phage 15/P, Pseudomonas phage 119x, and E. coli T4 phage) as well as purified T4 phage on the course of murine collagen-induced arthritis (CIA), commonly used as an animal model of rheumatoid arthritis. Intraperitoneal application of phage lysates or purified T4 phage did not aggravate the course of autoimmune joint disease. Moreover, although endotoxins are known to potentiate CIA, the systemic administration of phage lysate of Pseudomonas aeruginosa, which contains debris of this Gram-negative bacillus, did not significantly influence CIA although the sonicate of the corresponding bacterial strain did. Interestingly, a purified T4 phage revealed some anti-inflammatory activity when applied under the therapeutic scheme. Our preliminary results do not suggest that phages may aggravate the symptoms of rheumatoid arthritis. In contrast T4 phage may even exert an immunosuppressive effect.

Bacteriophages targeting intestinal epithelial cells: a potential novel form of immunotherapy

In addition to their established role as a physical barrier to invading pathogens and other harmful agents, intestinal epithelial cells (IEC) are actively involved in local immune reactions. In the past years, evidence has accumulated suggesting the role of IEC in the immunopathology of intestinal inflammatory disorders (IBD). Recent advances in research on bacteriophages strongly suggest that—in addition to their established antibacterial activity—they have immunomodulating properties that are potentially useful in the clinic. We suggest that these immunomodulating phage activities targeting IEC may open novel treatment perspectives in disorders of the alimentary tract, particularly IBD.

Antibody Production in Response to Staphylococcal MS-1 Phage Cocktail in Patients Undergoing Phage Therapy

In this study, we investigated the humoral immune response (through the release of IgG, IgA, and IgM antiphage antibodies) to a staphylococcal phage cocktail in patients undergoing experimental phage therapy at the Phage Therapy Unit, Medical Center of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy in Wrocław, Poland. We also evaluated whether occurring antiphage antibodies had neutralizing properties toward applied phages (K rate). Among 20 examined patients receiving the MS-1 phage cocktail orally and/or locally, the majority did not show a noticeably higher level of antiphage antibodies in their sera during phage administration. Even in those individual cases with an increased immune response, mostly by induction of IgG and IgM, the presence of antiphage antibodies did not translate into unsatisfactory clinical results of phage therapy. On the other hand, a negative outcome of the treatment occurred in some patients who showed relatively weak production of antiphage antibodies before and during treatment. This may imply that possible induction of antiphage antibodies is not an obstacle to the implementation of phage therapy and support our assumption that the outcome of the phage treatment does not primarily depend on the appearance of antiphage antibodies in sera of patients during therapy. These conclusions are in line with our previous findings. The confirmation of this thesis is of great interest as regards the efficacy of phage therapy in humans.