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.

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.

Safety Studies of Pneumococcal Endolysins Cpl-1 and Pal.

Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 µM of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies.

Two novel temperate bacteriophages infecting Streptococcus pyogenes: Their genomes, morphology and stability.

Only 3% of phage genomes in NCBI nucleotide database represent phages that are active against Streptococcus sp. With the aim to increase general awareness of phage diversity, we isolated two bacteriophages, Str01 and Str03, active against health-threatening Group A Streptococcus (GAS). Both phages are members of the Siphoviridae, but their analysis revealed that Str01 and Str03 do not belong to any known genus. We identified their structural proteins based on LC-ESI29 MS/MS and list their basic thermal stability and physico-chemical features including optimum pH. Annotated genomic sequences of the phages are deposited in GenBank (NCBI accession numbers KY349816 and KY363359, respectively).

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.

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.

Bacteriophages displaying anticancer peptides in combined antibacterial and anticancer treatment.

AIMS: Novel anticancer strategies have employed bacteriophages as drug carriers and display platforms for anticancer agents; however, bacteriophage-based platforms maintain their natural antibacterial activity. This study provides the assessment of combined anticancer (engineered) and antibacterial (natural) phage activity in therapies. MATERIALS & METHODS: An in vivo BALB/c mouse model of 4T1 tumor growth accompanied by surgical wound infection was applied. The wounds were located in the areas of tumors. Bacteriophages (T4) were modified with anticancer Tyr-Ile-Gly-Ser-Arg (YIGSR) peptides by phage display and injected intraperitoneally. RESULTS & CONCLUSION: Tumor growth was decreased in mice treated with YIGSR-displaying phages. The acuteness of wounds, bacterial load and inflammatory markers in phages-treated mice were markedly decreased. Thus, engineered bacteriophages combine antibacterial and anticancer activity.

Immunogenicity studies of proteins forming the T4 phage head surface.

UNLABELLED: Advances in phage therapy and novel applications of phages in biotechnology encourage interest in phage impact on human and animal immunity. Here we present comparative studies of immunogenic properties of T4 phage head surface proteins gp23*, gp24*, Hoc, and Soc, both as elements of the phage capsid and as isolated agents. Studies comprise evaluation of specific antibodies in the human population, analysis of the proteins' impact on the primary and secondary responses in mice, and the effect of specific antibodies on phage antibacterial activity in vitro and in vivo in mice. In humans, natural antibodies specific to T4-like phages were abundant (81% of investigated sera). Among those, significantly elevated levels of IgG antibodies only against major head protein (gp23*) were found, which probably reflected cross-reactions of T4 with antibodies induced by other T4-like phages. Both IgM and IgG antibodies were induced mostly by gp23* and Hoc, while weak (gp24*) and very weak (Soc) reactivities of other head proteins were noticed. Thus, T4 head proteins that markedly contribute to immunological memory to the phage are highly antigenic outer capsid protein (Hoc) and major capsid protein (gp23*). Specific anti-gp23* and anti-Hoc antibodies substantially decreased T4 phage activity in vitro and to some extent in vivo. Cooperating with antibodies, the immune complement system also contributed to annihilating phages. IMPORTANCE: Current descriptions of phage immunogenicity and its biological consequences are still vague and incomplete; thus, the central problem of this work is timely and may have strong practical implications. Here is presented the very first description of the contribution of bacteriophage proteins to immunological memory of the phage. Understanding of interactions between phages and mammalian immunology may help in biotechnological adaptations of phages for therapeutic requirements as well as for better appreciation of phage ecology and their role in the biosphere.

Molecular imaging of T4 phage in mammalian tissues and cells.

Advances in phage therapy encourage scientific interest in interactions of phages with human and animal organisms. This has created a need for developing tools that facilitate studies of phage circulation and deposition in tissues and cells. Here we propose a new green fluorescent protein (GFP)-based method for T4 phage molecular imaging in living systems. The method employs decoration of a phage capsid with GFP fused to the N-terminus of Hoc protein by in vivo phage display. Fluorescent phages were positively assessed as regards their applicability for detection inside living mammalian cells (by phagocytosis) and tissues (filtering and retention by lymph nodes and spleen). Molecular imaging provides innovative techniques that have brought substantial progress in life sciences. We propose it as a useful tool for studies of phage biology.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.