Immunogenic epitope scanning in bacteriolytic enzymes Pal and Cpl-1 and engineering Pal to escape antibody responses.

Bacteriolytic enzymes are promising antibacterial agents, but they can cause a typical immune response in vivo. In this study, we used a targeted modification method for two antibacterial endolysins, Pal and Cpl-1. We identified the key immunogenic amino acids, and designed and tested new, bacteriolytic variants with altered immunogenicity. One new variant of Pal (257-259 MKS → TFG) demonstrated decreased immunogenicity while a similar mutant (257-259 MKS → TFK) demonstrated increased immunogenicity. A third variant (280-282 DKP → GGA) demonstrated significantly increased antibacterial activity and it was not cross-neutralized by antibodies induced by the wild-type enzyme. We propose this variant as a new engineered endolysin with increased antibacterial activity that is capable of escaping cross-neutralization by antibodies induced by wild-type Pal. We show that efficient antibacterial enzymes that avoid cross-neutralization by IgG can be developed by epitope scanning, in silico design, and substitutions of identified key amino acids with a high rate of success. Importantly, this universal approach can be applied to many proteins beyond endolysins and has the potential for design of numerous biological drugs.

Evolution of the T4 phage virion is driven by selection pressure from non-bacterial factors.

Bacteriophages colonize animal and human bodies, propagating on sensitive bacteria that are symbionts, commensals, or pathogens of animals and humans. T4-like phages are dependent on abundant symbionts such as Escherichia coli, commonly present in animal and human gastrointestinal (GI) tracts. Bacteriophage T4 is one of the most complex viruses, and its intricate structure, particularly the capsid head protecting the phage genome, likely contributes substantially to the overall phage fitness in diverse environments. We investigated how individual head proteins-gp24, Hoc, and Soc-affect T4 phage survival under pressure from non-bacterial factors. We constructed a panel of T4 phage variants defective in these structural proteins: T4∆Soc, T4∆24byp24, T4∆Hoc∆Soc, T4∆Hoc∆24byp24, T4∆Soc∆24byp24, and T4∆Hoc∆Soc∆24byp24 (byp = bypass). These variants were investigated for their sensitivity to selected environmental conditions relevant to the microenvironment of the GI tract, including pH, temperature, and digestive enzymes. The simple and "primitive" structure of the phage capsid (∆24byp24) was significantly less stable at low pH and more sensitive to inactivation by digestive enzymes, and the simultaneous lack of gp24 and Soc resulted in a notable decrease in phage activity at 37°C. Gp24 was also found to be highly resistant to thermal and chemical denaturation. Thus, gp24, which was acquired relatively late in evolution, seems to play a key role in T4 withstanding environmental conditions, including those related to the animal/human GI tract, and Soc is a molecular glue that enhances this protective effect. IMPORTANCE Bacteriophages are important components of animal and human microbiota, particularly in the gastrointestinal tract, where they dominate the viral community and contribute to shaping microbial balance. However, interactions with bacterial hosts are not the only element of the equation in phage survival-phages inhabiting the GI tract are constantly exposed to increased temperature, pH fluctuations, or digestive enzymes, which raises the question of whether and how the complex structure of phage capsids contributes to their persistence in the specific microenvironment of human/animal bodies. Here we address this phage-centric perspective, identifying the role of individual head proteins in T4 phage survival in GI tract conditions. The selection pressure driving the evolution of T4-like phages could have come from the external environment that affects phage virions with increased temperature and variable pH; it is possible that in the local microenvironment along the GI tract, the phage benefits from stability-protecting proteins.

Experimental Identification of Cross-Reacting IgG Hotspots to Predict Existing Immunity Evasion of SARS-CoV-2 Variants by a New Biotechnological Application of Phage Display.

Multiple pathogens are competing against the human immune response, leading to outbreaks that are increasingly difficult to control. For example, the SARS-CoV-2 virus continually evolves, giving rise to new variants. The ability to evade the immune system is a crucial factor contributing to the spread of these variants within the human population. With the continuous emergence of new variants, it is challenging to comprehend all the possible combinations of previous infections, various vaccination types, and potential exposure to new variants in an individual patient. Rather than conducting variant-to-variant comparisons, an efficient approach could involve identifying key protein regions associated with the immune evasion of existing immunity against the virus. In this study, we propose a new biotechnological application of bacteriophages, the phage display platform for experimental identification of regions (linear epitopes) that may function as cross-reacting IgG hotspots in SARS-CoV-2 structural proteins. A total of 34,949 epitopes derived from genomes of all SARS-CoV-2 variants deposited prior to our library design were tested in a single assay. Cross-reacting IgG hotspots are protein regions frequently recognized by cross-reacting antibodies in many variants. The assay facilitated the one-step identification of immunogenic regions of proteins that effectively induced specific IgG in SARS-CoV-2-infected patients. We identified four regions demonstrating both significant immunogenicity and the activity of a cross-reacting IgG hotspot in protein S (located at NTD, RBD, HR1, and HR2/TM domains) and two such regions in protein N (at 197-280 and 358-419 aa positions). This novel method for identifying cross-reacting IgG hotspots holds promise for informing vaccine design and serological diagnostics for COVID-19 and other infectious diseases.

Dynamics of anti-SARS-CoV-2 seroconversion in individual patients and at the population level.

The immune response and specific antibody production in COVID-19 are among the key factors that determine both prognostics for individual patients and the global perspective for controlling the pandemics. So called "dark figure", that is, a part of population that has been infected but not registered by the health care system, make it difficult to estimate herd immunity and to predict pandemic trajectories. Here we present a follow up study of population screening for hidden herd immunity to SARS-CoV-2 in individuals who had never been positively diagnosed against SARS-CoV-2; the first screening was in May 2021, and the follow up in December 2021. We found that specific antibodies targeting SARS-CoV-2 detected in May as the "dark figure" cannot be considered important 7 months later due to their significant drop. On the other hand, among participants who at the first screening were negative for anti-SARS-CoV-2 IgG, and who have never been diagnosed for SARS-CoV-2 infection nor vaccinated, 26% were found positive for anti-SARS-CoV-2 IgG. This can be attributed to of the "dark figure" of the recent, fourth wave of the pandemic that occurred in Poland shortly before the study in December. Participants who were vaccinated between May and December demonstrated however higher levels of antibodies, than those who undergone mild or asymptomatic (thus unregistered) infection. Only 7% of these vaccinated participants demonstrated antibodies that resulted from infection (anti-NCP). The highest levels of protection were observed in the group that had been infected with SARS-CoV-2 before May 2021 and also fully vaccinated between May and December. These observations demonstrate that the hidden fraction of herd immunity is considerable, however its potential to suppress the pandemics is limited, highlighting the key role of vaccinations.

Correlation between COVID-19 severity and previous exposure of patients to Borrelia spp.

Predictors for the risk of severe COVID-19 are crucial for patient care and control of the disease. Other infectious diseases as potential comorbidities in SARS-CoV-2 infection are still poorly understood. Here we identify association between the course of COVID-19 and Lyme disease (borreliosis), caused by Borrelia burgdorferi transmitted to humans by ticks. Exposure to Borrelia was identified by multi-antigenic (19 antigens) serological testing of patients: severe COVID-19 (hospitalized), asymptomatic to mild COVID-19 (home treated or not aware of being infected), and not infected with SARS-CoV-2. Increased levels of Borrelia-specific IgGs strongly correlated with COVID-19 severity and risk of hospitalization. This suggests that a history of tick bites and related infections may contribute to the risks in COVID-19. Though mechanisms of this link is not clear yet, screening for antibodies targeting Borrelia may help accurately assess the odds of hospitalization for SARS-CoV-2 infected patients, supporting efforts for efficient control of COVID-19.

Hidden fraction of Polish population immune to SARS-CoV-2 in May 2021.

Population immunity (herd immunity) to SARS-CoV-2 derives from two sources: vaccinations or cases of infection with the virus. Infections can be diagnosed as COVID-19 and registered, or they can be asymptomatic, oligosymptomatic, or even full-blown but undiagnosed and unregistered when patients recovered at home. Estimation of population immunity to SARS-CoV-2 is difficult and remains a subject of speculations. Here we present a population screening for SARS-CoV-2 specific IgG and IgA antibodies in Polish citizens (N = 501) who had never been positively diagnosed with or vaccinated against SARS-CoV-2. Serum samples were collected in Wroclaw (Lower Silesia) on 15th and 22nd May 2021. Sera from hospitalized COVID-19 patients (N = 22) or from vaccinated citizens (N = 14) served as positive controls. Sera were tested with Microblot-Array COVID-19 IgG and IgA (quantitative) that contain specific SARS-CoV-2 antigens: NCP, RBD, Spike S2, E, ACE2, PLPro protein, and antigens for exclusion cross-reactivity with other coronaviruses: MERS-CoV, SARS-CoV, HCoV 229E Np, HCoV NL63 Np. Within the investigated population of healthy individuals who had never been positively diagnosed with or vaccinated against SARS-CoV-2, we found that 35.5% (178 out of 501) were positive for SARS-CoV-2-specific IgG and 52.3% (262 out of 501) were positive for SARS-CoV-2-specific IgA; 21.2% of the investigated population developed virus-specific IgG or IgA while being asymptomatic. Anti-RBD IgG, which represents virus-neutralizing potential, was found in 25.6% of individuals (128 out of 501). These patients, though positive for anti-SARS-CoV-2 antibodies, cannot be identified in the public health system as convalescents due to undiagnosed infections, and they are considered unaffected by SARS-CoV-2. Their contribution to population immunity against COVID-19 should however be considered in predictions and modeling of the COVID-19 pandemic. Of note, the majority of the investigated population still lacked anti-RBD IgG protection (74.4%); thus vaccination against COVID-19 is still of the most importance for controlling the pandemic.

Peptide Charge Derivatization as a Tool for Early Detection of Preeclampsia by Mass Spectrometry-A Comparison with the ELISA Test.

Early detection of any preeclampsia biomarkers may lower the risk of mortality, both for a mother and a child. Our study focuses on techniques for preeclampsia biomarker identification by comparing the results of a method using liquid chromatography mass spectrometry in multiple reaction monitoring mode (LC-MS/MS) with those by the enzyme-linked immunosorbent assay (ELISA) test, as well as by comparing the obtained results with clinical data. In the proposed LC-MS/MS method a tryptic digest peptide charge derivatization strategy was used as a tool for sensitive detection of podocin, i.e., a previously discovered preeclampsia biomarker present in urine samples from pregnant women. Urine samples from pregnant women with diagnosed preeclampsia were collected at different stages of pregnancy and from healthy subjects, and then were analyzed by ELISA test and the proposed method with LC-MS/MS. Charge derivatization of the ε amino group of C-terminal lysine residues in tryptic digests by 2,4,6-triphenylpyrylium salt was performed to increase the ionization efficiency in the LC-MS/MS mode. Podocin was identified at the early stage of pregnancy, while its detection using an ELISA test was not possible. The protocol for urine sample preparation was optimized. Our results show that the proposed method by LC-MS/MS in combination with peptide charge derivatization, provides an ultrasensitive tool for diagnosis of preeclampsia, and provides earlier detection than a clinical diagnosis or ELISA test. The proposed solution may revolutionize medical diagnostics.

DNA Dye Sytox Green in Detection of Bacteriolytic Activity: High Speed, Precision and Sensitivity Demonstrated With Endolysins.

Introduction: Increasing number of deaths from multi-drug resistant bacterial infections has caused both the World Health Organization and the Centers for Disease Control and Prevention to repeatedly call for development of new, non-traditional antibacterial treatments. Antimicrobial enzymes, including those derived from bacteriophages, known as endolysins or enzybiotics, are considered promising solutions among the emerging therapies. These naturally occurring proteins specifically destroy bacterial cell walls (peptidoglycan) and as such, are capable of killing several logs of bacteria within minutes. Some endolysins cause lysis of a wide range of susceptible bacteria, including both Gram-positive and Gram-negative organisms, whereas other endolysins are species- or even strain-specific. To make wide use of endolysins as antibacterial agents, some basic research issues remain to be clarified or addressed. Currently available methods for testing endolysin kinetics are indirect, require large numbers of bacteria, long incubation times and are affected by technical problems or limited reproducibility. Also, available methods are focused more on enzymatic activity rather than killing efficiency which is more relevant from a medical perspective. Results: We show a novel application of a DNA dye, SYTOX Green. It can be applied in comprehensive, real-time and rapid measurement of killing efficiency, lytic activity, and susceptibility of a bacterial population to lytic enzymes. Use of DNA dyes shows improved reaction times, higher sensitivity in low concentrations of bacteria, and independence of bacterial growth. Our data show high precision in lytic activity and enzyme efficiency measurements. This solution opens the way to the development of new, high throughput, precise measurements and tests in variety of conditions, thus unlocking new possibilities in development of novel antimicrobials and analysis of bacterial samples.

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.

Circulation of Fluorescently Labelled Phage in a Murine Model.

Interactions between bacteriophages and mammals strongly affect possible applications of bacteriophages. This has created a need for tools that facilitate studies of phage circulation and deposition in tissues. Here, we propose red fluorescent protein (RFP)-labelled E. coli lytic phages as a new tool for the investigation of phage interactions with cells and tissues. The interaction of RFP-labelled phages with living eukaryotic cells (macrophages) was visualized after 20 min of co-incubation. RFP-labeled phages were applied in a murine model of phage circulation in vivo. Phages administered by three different routes (intravenously, orally, rectally) were detected through the course of time. The intravenous route of administration was the most efficient for phage delivery to multiple body compartments: 20 min after administration, virions were detected in lymph nodes, lungs, and liver; 30 min after administration, they were detectable in muscles; and 1 h after administration, phages were detected in spleen and lymph nodes. Oral and rectal administration of RFP-labelled phages allowed for their detection in the gastrointestinal (GI) tract only.

Endocytosis in cellular uptake of drug delivery vectors: Molecular aspects in drug development.

Drug delivery vectors are widely applied to increase drug efficacy while reducing the side effects and potential toxicity of a drug. They allow for patient-tailored therapy, dose titration, and therapeutic drug monitoring. A major part of drug delivery systems makes use of large nanocarriers: liposomes or virus-like particles (VLPs). These systems allow for a relatively large amount of cargo with good stability of vectors, and they offer multiple options for targeting vectors in vivo. Here we discuss endocytic pathways that are available for drug delivery by large nanocarriers. We focus on molecular aspects of the process, including an overview of potential molecular targets for studies of drug delivery vectors and for future solutions allowing targeted drug delivery.

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.

Induction of Phage-Specific Antibodies by Two Therapeutic Staphylococcal Bacteriophages Administered per os.

In therapeutic phage applications oral administration is a common and well-accepted delivery route. Phages applied per os may elicit a specific humoral response, which may in turn affect phage activity. We present specific anti-phage antibody induction in mice receiving therapeutic staphylococcal bacteriophage A3R or 676Z in drinking water. The schedule comprised: (1) primary exposure to phages for 100 days, followed by (2) diet without phage for 120 days, and (3) secondary exposure to the same phage for 44 days. Both phages induced specific antibodies in blood (IgM, IgG, IgA), even though poor to ineffective translocation of the phages to blood was observed. IgM reached a maximum on day 22, IgG increased from day 22 until the end of the experiment. Specific IgA in the blood and in the gut were induced simultaneously within about 2 months; the IgA level gradually decreased when phage was removed from the diet. Importantly, phage-specific IgA was the limiting factor for phage activity in the gastrointestinal tract. Multicopy proteins (major capsid protein and tail morphogenetic protein H) contributed significantly to phage immunogenicity (IgG), while the baseplate protein gpORF096 did not induce a significant response. Microbiome composition assessment by next-generation sequencing (NGS) revealed that no important changes correlated with phage treatment.

Bacteriophages engineered to display foreign peptides may become short-circulating phages.

Bacteriophages draw scientific attention in medicine and biotechnology, including phage engineering, widely used to shape biological properties of bacteriophages. We developed engineered T4-derived bacteriophages presenting seven types of tissue-homing peptides. We evaluated phage accumulation in targeted tissues, spleen, liver and phage circulation in blood (in mice). Contrary to expectations, accumulation of engineered bacteriophages in targeted organs was not observed, but instead, three engineered phages achieved tissue titres up to 2 orders of magnitude lower than unmodified T4. This correlated with impaired survival of these phages in the circulation. Thus, engineering of T4 phage resulted in the short-circulating phage phenotype. We found that the complement system inactivated engineered phages significantly more strongly than unmodified T4, while no significant differences in phages' susceptibility to phagocytosis or immunogenicity were found. The short-circulating phage phenotype of the engineered phages suggests that natural phages, at least those propagating on commensal bacteria of animals and humans, are naturally optimized to escape rapid neutralization by the immune system. In this way, phages remain active for longer when inside mammalian bodies, thus increasing their chance of propagating on commensal bacteria. The effect of phage engineering on phage pharmacokinetics should be considered in phage design for medical purposes.

Interaction of Bacteriophages with Mammalian Cells.

Natural bacteriophages (present in the microbiome) and those applied as therapeutic agents may interact with mammalian cells and tissues. Adhesion interactions may define bacteriophage pharmacokinetics and resulting efficiency of bacteriophage agents in therapeutic applications by shaping bacteriophage homing to tissues and organs. Here we propose protocols for testing direct adhesion of bacteriophages or bacteriophage proteins to mammalian cells (in vitro). We further propose an animal model for investigation of accumulation/homing of bacteriophages in tissues (in vivo).

Real-Time qPCR as a Method for Detection of Antibody-Neutralized Phage Particles.

The most common method for phage quantitation is the plaque assay, which relies on phage ability to infect bacteria. However, non-infective phage particles may preserve other biological properties; specifically, they may enter interactions with the immune system of animals and humans. Here, we demonstrate real-time quantitative polymerase chain reaction (qPCR) detection of bacteriophages as an alternative to the plaque assay. The closely related staphylococcal bacteriophages A3R and 676Z and the coliphage T4 were used as model phages. They were tested in vivo in mice, ex vivo in human sera, and on plastic surfaces designed for ELISAs. T4 phage was injected intravenously into pre-immunized mice. The phage was completely neutralized by specific antibodies within 5 h (0 pfu/ml of serum, as determined by the plaque assay), but it was still detected by qPCR in the amount of approximately 107 pfu/ml of serum. This demonstrates a substantial timelapse between "microbiological disappearance" and true clearance of phage particles from the circulation. In human sera ex vivo, qPCR was also able to detect neutralized phage particles that were not detected by the standard plaque assay. The investigated bacteriophages differed considerably in their ability to immobilize on plastic surfaces: this difference was greater than one order of magnitude, as shown by qPCR of phage recovered from plastic plates. The ELISA did not detect differences in phage binding to plates. Major limitations of qPCR are possible inhibitors of the PCR reaction or free phage DNA, which need to be considered in procedures of phage sample preparation for qPCR testing. We propose that phage pharmacokinetic and pharmacodynamic studies should not rely merely on detection of antibacterial activity of a phage. Real-time qPCR can be an alternative for phage detection, especially in immunological studies of bacteriophages. It can also be useful for studies of phage-based drug nanocarriers or biosensors.

Spectroscopic, structural and in vitro cytotoxicity evaluation of luminescent, lanthanide doped core@shell nanomaterials GdVO4:Eu(3+)5%@SiO2@NH2.

The luminescent GdVO4:Eu(3+)5%@SiO2@NH2 core@shell nanomaterials were obtained via co-precipitation method, followed by hydrolysis and co-condensation of silane derivatives: tetraethyl orthosilicate and 3-aminopropyltriethoxysilane. Their effect on human erythrocytes sedimentation and on proliferation of human lung microvascular endothelial cells was examined and discussed. The luminescent nanoparticles were synthesized in the presence of polyacrylic acid or glycerin in order to minimalize the agglomeration and excessive growth of nanostructures. Surface coating with amine functionalized silica shell improved their biocompatibility, facilitated further organic conjugation and protected the internal core. Magnetic measurements revealed an enhanced T1-relaxivity for the synthesized GdVO4:Eu(3+)5% nanostructures. Structure, morphology and average grain size of the obtained nanomaterials were determined by X-ray diffraction, transmission electron microscopy and dynamic light scattering analysis. The qualitative elemental composition of the nanomaterials was established using energy-dispersive X-ray spectroscopy. The spectroscopic characteristic of red emitting core@shell nanophosphors was completed by measuring luminescence spectra and decays. The emission spectra revealed characteristic bands of Eu(3+) ions related to the transitions (5)D0-(7)F0,1,2,3,4 and (5)D1-(7)F1. The luminescence lifetimes consisted of two components, associated with the presence of Eu(3+) ions located at the surface of the crystallites and in the bulk.

T4 Phage Tail Adhesin Gp12 Counteracts LPS-Induced Inflammation In Vivo.

Bacteriophages that infect Gram-negative bacteria often bind to the bacterial surface by interaction of specific proteins with lipopolysaccharide (LPS). Short tail fiber proteins (tail adhesin, gp12) mediate adsorption of T4-like bacteriophages to Escherichia coli, binding surface proteins or LPS. Produced as a recombinant protein, gp12 retains its ability to bind LPS. Since LPS is able to exert a major impact on the immune response in animals and in humans, we have tested LPS-binding phage protein gp12 as a potential modulator of the LPS-induced immune response. We have produced tail adhesin gp12 in a bacterial expression system and confirmed its ability to form trimers and to bind LPS in vitro by dynamic light scattering. This product had no negative effect on mammalian cell proliferation in vitro. Further, no harmful effects of this protein were observed in mice. Thus, gp12 was used in combination with LPS in a murine model, and it decreased the inflammatory response to LPS in vivo, as assessed by serum levels of cytokines IL-1 alpha and IL-6 and by histopathological analysis of spleen, liver, kidney and lungs. Thus, in future studies gp12 may be considered as a potential tool for modulating and specifically for counteracting LPS-related physiological effects in vivo.

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.

Oral Application of T4 Phage Induces Weak Antibody Production in the Gut and in the Blood.

A specific humoral response to bacteriophages may follow phage application for medical purposes, and it may further determine the success or failure of the approach itself. We present a long-term study of antibody induction in mice by T4 phage applied per os: 100 days of phage treatment followed by 112 days without the phage, and subsequent second application of phage up to day 240. Serum and gut antibodies (IgM, IgG, secretory IgA) were analyzed in relation to microbiological status of the animals. T4 phage applied orally induced anti-phage antibodies when the exposure was long enough (IgG day 36, IgA day 79); the effect was related to high dosage. Termination of phage treatment resulted in a decrease of IgA again to insignificant levels. Second administration of phage induces secretory IgA sooner than that induced by the first administrations. Increased IgA level antagonized gut transit of active phage. Phage resistant E. coli dominated gut flora very late, on day 92. Thus, the immunological response emerges as a major factor determining phage survival in the gut. Phage proteins Hoc and gp12 were identified as highly immunogenic. A low response to exemplary foreign antigens (from Ebola virus) presented on Hoc was observed, which suggests that phage platforms can be used in oral vaccine design.