Detection and Level Evaluation of Antibodies Specific to Environmental Bacteriophage I11mO19 and Related Coliphages in Non-Immunized Human Sera.

Bacteriophages (phages) are viruses infecting bacteria. They are widely present in the environment, food, and normal microflora. The human microbiome is a mutually interdependent network of bacteria, bacteriophages, and human cells. The stability of these tri-kingdom interactions may be essential for maintaining immunologic and metabolic health. Phages, as with each other's antigens, may evoke an immune response during a human's lifetime and induce specific antibody generation. In this manuscript, we labeled these antibodies as naturally generated. Naturally generated antibodies may be one of the most important factors limiting the efficacy of phage therapy. Herein, we attempted to determine the physiological level of these antibodies specific to a population bacteriophage named I11mO19 in human sera, using an ELISA-based assay. First, we purified the phage particles and assessed the immunoreactivity of phage proteins. Then, affinity chromatography was performed on columns with immobilized phage proteins to obtain a fraction of human polyclonal anti-phage antibodies. These antibodies were used as a reference to elaborate an immunoenzymatic test that was used to determine the level of natural anti-phage antibodies. We estimated the average level of anti-I11mO19 phage antibodies at 190 µg per one milliliter of human serum. However, immunoblotting revealed that cross-reactivity occurs between some proteins of I11mO19 and two other coliphages: T4 and ΦK1E. The antigens probably share common epitopes, suggesting that the determined level of anti-I11mO19 phage might be overestimated and reflects a group of antibodies reactive to a broad range of other E. coli phages. Anti-I11mO19 antibodies did not react with Pseudomonas bacteriophage F8, confirming specificity to the coliphage group. In this work, we wanted to show whether it is possible to determine the presence and level of anti-phage antibodies in nontargeted-immunized sera, using an immunoenzymatic assay. The conclusion is that it is possible, and specific antibodies can be determined. However, the specificity refers to a broader coliphage group of phages, not only the single phage strain.

[Approaches to the estimation of some parameters of humoral and cellular immune response to bacteriophages.]

The aim of the study was to develop some approaches to evaluate the basic parameters of the humoral and cellular immune response to a bacteriophage, taking into account the multifactorial aspects of its interaction with both the pathogen and the macroorganism. The necessary reagents were obtained and a line of diagnostic ELISA test systems was designed to allow semi-quantitative assessment of the anti-bacteriophage IgG-antibody level in serum or other biological human fluids, as well as in preparations obtained from human blood. The need for neutralization reaction to determine the effect of detected antibodies on phage activity against a target bacterium has been proven. Testing the approaches used in the investigation of patients' blood sera showed that antibodies to bacteriophages synthesized during phage therapy are not always neutralizing. Also approaches have been developed to evaluate cell immunity reactions to bacteriophage namely to identify T-lymphocytes (T-helpers and cytotoxic lymphocytes) that can be activated in the presence of the phage under study (by expressing the early activation marker (CD69) and by the ability to produce IFNγ). Approbation of the technique in the study of lymphcytes in patients during phage therapy showed the presence of activated cells by both the CD69 expression and IFNγ production, the dynamics of which depended on the timing and frequency of therapy. The appearance of neutralizing anti-phage antibodies and corresponding activated T-lymphocytes should be taken into account in phage therapy, the effectiveness of which can directly depend not only on the activity of the phage against the target bacterium, but also on the response of the patient's immune system to the bacteriophage.

Phage Therapy: What Have We Learned?

In this article we explain how current events in the field of phage therapy may positively influence its future development. We discuss the shift in position of the authorities, academia, media, non-governmental organizations, regulatory agencies, patients, and doctors which could enable further advances in the research and application of the therapy. In addition, we discuss methods to obtain optimal phage preparations and suggest the potential of novel applications of phage therapy extending beyond its anti-bacterial action.

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.