Therapeutic phages are being employed for vaccination and treatment of cancer and bacterial infections. Their natural immunogenicity triggers intertwined interactions with innate and adaptive immune cells that might influence therapy. Phage- and bactierial-derived pathogen-associated molecular patterns released after bacterial lysis have been proposed to stimulate local innate immune responses, which could promote antitumor immunity or bacterial clearance. Conversely, immunogenicity of phages induces phage-specific humoral memory, which can hamper therapeutic success. This review outlines the current knowledge on the different types of immune responses elicited by phages and their potential benefits and adverse side effects, when applied therapeutically. This review further summarizes the knowledge gaps and defines the key immunological questions that need to be addressed regarding the clinical application of antibacterial phage therapy.

Increased antibiotic resistance and subsequent therapeutic failure of antibiotics have fueled the discussion on potential alternatives to antibiotics for treatment of bacterial infections. Many of these alternative approaches are based on development of biologicals. These include 1) passive immunization with Ig preparations enriched for pathogen-specific Abs or mAbs directed at known pathogens, 2) induction of a pathogen-specific memory response by vaccination, 3) targeted modulation of the host immune response via immune stimulatory treatments that facilitate host-mediated clearance of the extracellular and/or intracellular infecting microbe, and 4) elimination of bacteria by lytic phages. With the exception of bacteriophage therapy, these concepts are currently referred to as host-directed therapies (1). Among biologicals, phage therapy is currently experiencing a renaissance because lytic phages targeting antibiotic-resistant bacterial pathogens might represent an efficient therapy if development is driven to match the clinical needs and meet regulatory requirements (2). However, despite concerted efforts from academia, regulators, and biotech companies, medicinal products for phage therapy are not available in countries with regulatory requirements demanding a high level of clinical evidence. In this context, uncertainties in regard to the impact of the immune response on efficacy and safety are important obstacles to defining the target product profiles and obtaining regulatory approval.

Despite the use and effectiveness of antibiotics, physicians are well aware of the role of the immune system in successful treatment of bacterial infections. Notably, an important principle of treatment with bacteriostatic antibiotics is that antibiotic control of bacterial proliferation and spread enables bacterial clearance by host immune cells. Furthermore, antibiotic-mediated damage of bacterial cells facilitates phagosomal lysis and release of pathogen-associated molecular patterns (PAMPs), which enhance bacterial clearance by activating the innate immune system (36). In cases of high bacterial burden, excess release of high amounts of endotoxin (i.e., LPS and other PAMPs) following administration of the antibiotic promotes a systemic inflammatory reaction that can require medical intervention (7).

Similarly to antibiotics, it has been suggested that activation of innate immunity by phage-mediated lysis of bacterial cells could contribute to the efficacy of phage therapy. By contrast, adverse effects of phage therapy have been attributed to toxicity of LPS contaminations in phage preparations (8, 9). However, unlike antibiotics, phages themselves are immunogenic microorganisms that can stimulate an adaptive immune response and bear the potential to interfere with repetitive treatments. Table I provides a summary and a comparison of the relevant characteristics of antibiotics and antibacterial phage therapeutics.

Table I.
Comparison of antibacterial therapies with antibiotics or lytic phages
AntibioticsLytic Phages
Mechanism of action Inhibition of cell wall synthesis, DNA replication, or protein synthesis Infection and subsequent lysis of bacteria 
Specificity (Usually) broad spectrum: Gram-negative or Gram-positive species or both Narrow spectrum: one or many individual strains within a bacterial species 
Vital microorganism No Yes (inactivation by heat or low pH) 
Innate immune stimulation No direct effect on innate immune cells Phages contain PAMPs such as DNA and RNA. 
Release of PAMPs upon loss of bacterial cell wall integrity Release of PAMPs upon bacterial cell lysis 
Ab induction No Yes (phages are complex biological organisms bearing immunogenic proteins) 
Half-time life Several hours up to 1 d Depending on host immunity and target species bioburden, hours to weeks 
Resistance development Natural resistance (target missing) Natural resistance (presence of nonsusceptible strains) 
Acquired resistance (accessory genomic elements encoding resistance mechanisms) Acquired resistance (selection of nonsusceptible strains based on CRISPR-Cas system, target modification, etc.) 
Resistance development upon exposure (mutations)  
Elimination of intracellular bacteria Possible with cell permeant antibiotics Questionable, possible by concomitant uptake of bacteria and phages 
AntibioticsLytic Phages
Mechanism of action Inhibition of cell wall synthesis, DNA replication, or protein synthesis Infection and subsequent lysis of bacteria 
Specificity (Usually) broad spectrum: Gram-negative or Gram-positive species or both Narrow spectrum: one or many individual strains within a bacterial species 
Vital microorganism No Yes (inactivation by heat or low pH) 
Innate immune stimulation No direct effect on innate immune cells Phages contain PAMPs such as DNA and RNA. 
Release of PAMPs upon loss of bacterial cell wall integrity Release of PAMPs upon bacterial cell lysis 
Ab induction No Yes (phages are complex biological organisms bearing immunogenic proteins) 
Half-time life Several hours up to 1 d Depending on host immunity and target species bioburden, hours to weeks 
Resistance development Natural resistance (target missing) Natural resistance (presence of nonsusceptible strains) 
Acquired resistance (accessory genomic elements encoding resistance mechanisms) Acquired resistance (selection of nonsusceptible strains based on CRISPR-Cas system, target modification, etc.) 
Resistance development upon exposure (mutations)  
Elimination of intracellular bacteria Possible with cell permeant antibiotics Questionable, possible by concomitant uptake of bacteria and phages 

CRISPR-Cas is a genome editing system found in bacteria and archaea that hinders integration of foreign DNA, characterized by clustered regularly interspaced short palindromic repeats (CRISPR) and endonuclease activity (Cas).

Moreover, recent mathematical modeling of phage–bacteria interactions suggests that the phage alone is unable to exterminate the whole bacterial population and that cooperation with the immune system is a prerequisite for successful phage therapy (10, 11). Considering the potential clinical applications for phage therapy, this would imply that either all or certain subgroups of immunosuppressed patients (e.g., the patients with highest need of antibacterial therapy) would be excluded from phage therapy, resulting in, overall, only little benefit in light of the desperate search for new antibacterial therapies targeting nosocomial infections. It was, therefore, an important purpose of this review to deliver an analysis of the available data in search of potential limitations for a broad indication of phage therapy that would be grounded in effects arising from phage–immune system interaction.

The overall aim of the present review is to provide an overview of the published data and the conclusions to be drawn in regard to the beneficial and the unfavorable immunological effects of therapeutic phages, with an emphasis on antibacterial phage therapy. Nevertheless, summarizing the data reveals their major limitation: generalization of findings obtained with a specific phage or in a specific model is often difficult because the criteria defining when extrapolation would be appropriate and permissible are unknown.

Phages live where their prey live (e.g., in unsterile areas of the body). They form part of the physiological mammalian microflora. In the human intestine, dsDNA and ssDNA phages infect Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, whereas RNA phages are thought to be ingested with food, and are present only transiently (12, 13). Furthermore, phages appear to adhere to the mucosal surfaces of diverse animals, thus reducing microbial colonization and pathology at these interfaces and providing a non–host-derived layer of immunity (14). It has further been suggested that lytic phages regulate the composition of the microbiome and support its diversity and resilience. Interaction of phages with host innate immune cells and epithelial cells, cytokine profiles, and anti-phage Abs might, in turn, control the phageome composition (12). This concept is new and exciting, but it remains to be investigated in much more detail. Similarly, concerns that phage therapy could alter the composition of the microbiome or that the preexisting immune response to natural phages could interfere with phage therapy need to be addressed in relevant models to clarify the in vivo relevancy of these hypotheses.

To initiate the production of Abs and generate a long-lasting memory response, phages have to be processed and presented to T cells by APCs. Therefore, it is important to understand how phages circumvent the epithelial barriers and reach the normally sterile lymphoid organs. A series of studies were performed to demonstrate the translocation of phages through the mucosal barriers in the living organism. Different routes of phage application (e.g., oral, intranasal, and i.p.) tested in different rodent models resulted in the rapid appearance of phages in the bloodstream and their accumulation in the kidneys, spleen, liver, and thymus (reviewed in Ref. 15).

Moreover, parental administration of phages in human subjects led to rapid distribution to the spleen and other organs, where phages persisted for more than 4 d (reviewed in Ref. 16). Following oral administration to human subjects, phages were detected in blood and urine samples (see Refs. 15 and 16 for review). However, the ability of phages to penetrate mucosal barriers remains not fully understood. Several hypotheses have been generated: 1) transcytosis of phage particles via epithelial cells; 2) circumvention of the epithelial barrier by a Trojan Horse mechanism, in which phages hide inside bacteria; 3) direct sampling of the luminal content by intestinal dendritic cells; and 4) translocation of phages through a damaged epithelial barrier (17, 18). The latter seems most convincing because the published data suggest that the intestinal barrier efficiently restricts the translocation of the naturally occurring phages into the bloodstream and tissues of healthy animals (19). Also, the descriptions of naturally occurring phagemia are scarce (16). In contrast, application of high doses of phages or antibiotics may lead to massive lysis of susceptible bacterial host cells with consecutive release of endotoxin, which triggers inflammation and may thus promote leakiness of the intestinal barrier (20). Additionally, the receptors responsible for translocation remain elusive (21), and the significance of phage adherence to mucin for transepithelial translocation needs to be elucidated (22). In regard to phage therapy, a better understanding of the processes involved in translocation might allow the distinction of patients at risk for the formation of systemic adaptive responses that target orally or topically administered phage preparations.

The main function of innate immune cells is the recognition and elimination of foreign material and, when appropriate and necessary, the mounting of an adaptive immune response against it. Neutrophils and granulocytes are critical components of the innate immunity and represent the frontline defense against tissue invasion by bacteria and viruses. Since the 1960s, it has been acknowledged that leukocytes are not only able to bind phages in a time-, concentration-, and temperature-dependent manner but also internalize and eventually eliminate the phages (reviewed in Ref. 23). Whether specific receptors are involved in phage endocytosis (the term phagocytosis is used for particles >500 nm) is not completely resolved, but recognition of the lysine–glycine–aspartic acid motif in the p26 capsid protein of the T4 phage by β3 integrins on phagocytes might be implicated in this process (16, 24). Fig. 1 visualizes the different cellular mechanisms that promote uptake of phages into macrophages and subsequent macrophage activation (Fig. 1).

FIGURE 1.

Phage–host immune cell interactions. The graph summarizes the multiple sites of interaction between phages (dark blue), bacteria (purple), and host macrophages (light blue). Upper panel, Phage-mediated lysis of bacterial cells. (A) Phages infect bacterial cells. (B) Phages replicate in the bacterial host. (C) Phages induce lysis of bacteria. (D) Phage-specific Abs (orange) block bacterial infection by phages. Lower panel, Phage-mediated activation of host macrophages in the presence (yellow endosomes) and absence (orange endosomes) of Abs (anti-phage Abs in orange; antibacterial Abs in red). (1) Phages expressing proteins that mediate host–phage interactions bind to macrophage surface receptors and activate macrophages. (2) Macrophages phagocytize extracellular bacterial cells and endocytose phages; bacterial and phage-derived PAMPs stimulate macrophage activity. (3) Macrophages phagocytize phage-infected bacterial cells; phage-derived PAMPs costimulate bacteria-induced macrophage activity. (4) After phage-induced bacterial lysis macrophages phagocytize bacterial debris and phages; bacterial and phage-derived PAMPs stimulate macrophage activity. (5) Macrophages phagocytize opsonized bacteria upon recognition of bacteria-specific Ig by Fc receptors (yellow); this enhances clearance of bacteria. (6) Phage-Ab complexes bind to Fc receptors (yellow) on macrophages; this triggers endocytosis and subsequent clearance of phages.

FIGURE 1.

Phage–host immune cell interactions. The graph summarizes the multiple sites of interaction between phages (dark blue), bacteria (purple), and host macrophages (light blue). Upper panel, Phage-mediated lysis of bacterial cells. (A) Phages infect bacterial cells. (B) Phages replicate in the bacterial host. (C) Phages induce lysis of bacteria. (D) Phage-specific Abs (orange) block bacterial infection by phages. Lower panel, Phage-mediated activation of host macrophages in the presence (yellow endosomes) and absence (orange endosomes) of Abs (anti-phage Abs in orange; antibacterial Abs in red). (1) Phages expressing proteins that mediate host–phage interactions bind to macrophage surface receptors and activate macrophages. (2) Macrophages phagocytize extracellular bacterial cells and endocytose phages; bacterial and phage-derived PAMPs stimulate macrophage activity. (3) Macrophages phagocytize phage-infected bacterial cells; phage-derived PAMPs costimulate bacteria-induced macrophage activity. (4) After phage-induced bacterial lysis macrophages phagocytize bacterial debris and phages; bacterial and phage-derived PAMPs stimulate macrophage activity. (5) Macrophages phagocytize opsonized bacteria upon recognition of bacteria-specific Ig by Fc receptors (yellow); this enhances clearance of bacteria. (6) Phage-Ab complexes bind to Fc receptors (yellow) on macrophages; this triggers endocytosis and subsequent clearance of phages.

Close modal

Once activated, macrophages produce an array of microbicide effectors and immunoregulatory cytokines that act in concert to eliminate the invasive agent and influence the course of the developing immune response. Lytic phages induce massive LPS release from Gram-negative bacteria; however, this does not exceed the amounts released under antibiotic treatment (25). Degradation of phages by polymorphonuclear cells and macrophages has been described for T2, φX174, λ, and P22 phages (see Ref. 26 for review). These studies demonstrate rapid phagocyte-mediated clearance of phages from organs, in particular the liver, where a rapid decline of highly infective phage titers was observed. Murine Kupffer cells, the resident liver phagocytes, were subsequently shown to be responsible for this process (27). Furthermore, in the spleen, high titers of phages were detectable irrespective of the route of phage immunization. Here, splenic macrophages were found to mediate clearance of phages, but they displayed an approximately four-times-slower kinetic than those observed with Kupffer cells (27).

Notably, the degradation process is also a prerequisite for Ag presentation and initiation of adaptive immune responses. It can thus be envisioned that improvement of phage therapy could include selection of phages with natural resistance to phagosomal degradation or rational design of recombinant phages to achieve this. This could avoid or delay induction of phage-specific adaptive immune responses and possibly prolong persistence of phages in immune-competent individuals.

A recent transcriptome analysis revealed that lysates from five different endotoxin-free phage preparations induced comparable cytokine signatures in human PBMCs (9). These cytokine profiles significantly differed from those induced by potentially contaminating LPS, and addition of LPS costimulated but did not alter the phage-induced cytokine profiles. Nevertheless, phage-mediated immune stimulation can most likely be attributed to the activation of pattern recognition receptors (PRR) by PAMPs present in phages. Phages contain genomic DNA and RNA (e.g., ligands for nucleic acid–sensing receptors, such as TLRs 7, 8, 9, and 13, RLR, and cytosolic DNA sensors) as well as compounds exerting TLR2 and TLR4 activity (28). Additionally to PAMPs, some phages express proteins that mediate interaction with mammalian host cells and thereby promote immune responses (29). Together with possible differences in endotoxin contamination levels, these virulence factors may account for the recently described differences in cytokine induction observed upon comparison of phages targeting Pseudomonas aeruginosa and Escherichia coli, respectively (3032).

The adjuvant properties attributed to the phages are being exploited for phage-based approaches to vaccination and therapy of cancer but may also influence other types of phage therapy (33, 34). TLR dependency has been observed in different types of phage therapies: 1) recruitment of tumor-associated macrophages and tumor regression was absent in MyD88-deficient mice treated with genetically engineered phages that target tumor cells in vivo (35, 36), 2) neutrophils were essential for successful treatment of pneumonia with anti–P. aeruginosa phages (11), 3) phage-mediated provision of Ag to dendritic cells and subsequent T cell activation were strongly impaired in the absence of MyD88 or TLR9 (37), and 4) vaccine responses to peptide-displaying phages were abrogated in the absence of MyD88 and altered in the absence of TLR9 (28). These findings support the hypothesis that interactions between phage-derived ligands and host PRR contribute to efficacy of phage therapies. This raises the question of the actual clinical impact of PRR activation and the concern that phage therapy could be less effective in patients with immune deficiencies that affect their innate immune responsiveness. To tackle this issue, clinical study protocols would need to include patients with different types of immune deficiencies.

Initial evidence for phage-mediated induction of adaptive immunity traces back to studies describing background neutralizing activity in sera and active immunization with well-described phages, such as ϕX174 and T4-like phages.

ϕX174.

The enterobacterial phage ϕX174 contains a small circular (+) ssDNA genome. The viral particles display specificity for LPS on the outer membrane of Gram-negative pathogens. Priming and boostering of the humoral immune response were initially demonstrated in guinea pigs and rabbits immunized with ϕX174 (3840). These experiments provided evidence for the presence of phage-specific short-lived IgM (19S) and memory responses (IgG/7S) with a long t1/2 life. In mice, immunization with this phage elicited neutralizing Ab responses in a dose-dependent manner (41). Of note, background levels of serum neutralizing activity were higher in conventional breeding than in germ-free mice. In humans, ϕX174 was used as a model Ag for induction of T cell–dependent Ab responses by diagnostic immunization with ϕX174 in patients with suspected common variable immune deficiency or hypogammaglobulinemia, HIV, or those who had undergone a splenectomy (4245). ϕX174-specific IgM and IgG responses were elicited in patients with intact B cell responses and absent or impaired in patients with B cell immunodeficiency or HIV. Furthermore, IgG responses to ϕX174 were absent in patients with defective Ig class switching, including splenectomized patients.

T4 and T4-like phages.

T2 and T4 phages have a dsDNA genome and target and lyse enterobacteria. Abs against the T2 phage were demonstrated in lymph node cultures from rats and in rabbit sera (46, 47). Binding of anti-T2 IgM and IgG to the head and tail of the phage was demonstrated with electron microscopy (48). Neutralizing Abs were found to react with the distal phage tail. Notably, a serum complement component was found to be necessary for neutralization of T2 but not ΦX174 in mice (49). Abs against T4-like phages were detected in 81% of tested human sera. IgG was directed against several phage head proteins and displayed neutralizing activity (29). In vivo experiments in mice revealed that immunization induced high titers of anti-Hoc Abs with neutralizing activity against T4; this correlated with a loss of T4-mediated protection against E. coli infection (29). Of note, the Hoc capsid glycoprotein was previously described as a highly immunogenic protein containing Ig-like domains but as nonessential in regard to infectivity of the phage (50, 51).

It was recently shown that heat-inactivated phages lose their immune stimulatory potential, whereas UV irradiation preserves immunogenicity (e.g., phage-induced Ab responses) elicited by live phage preparations (52). Interestingly enough, in 1961, Fishman described that a RNAse-sensitive substance in macrophage or T2 phage cocultures was capable of stimulating Ab synthesis (46). Further studies suggested a stimulatory role of phage-derived RNA in the induction of IFN (53). Because IFN-I triggers TLR7 expression in B cells, phage-derived RNA could directly contribute to B cell activation and the synthesis of anti-phage Abs (54). Well in line with these observations, in vivo experiments demonstrate that Ab production induced by the ssDNA phage M13 is TLR dependent (e.g., absent) in MyD88-deficient mice and decreased in TLR2-, TLR4-, and TLR7-deficient mice (28). Notably, in mice, M13 induced IgG2b/c and IgG3 responses but only weak IgG1 responses; the latter were increased in TLR9-deficient mice, thus confirming earlier observations that IgG1 is increased in the absence of TLR9 (55).

Based on these reports, phages deliver all necessary components for stimulation of naive B cells (56, 57): 1) immunogenic phage proteins enabling specific recognition via the BCR, 2) ligands for activation of costimulatory TLRs in B cells (e.g., nucleic acids), such as RNA derived from (viable) phages (danger signal), and 3) APC-mediated activation of helper T cells. Albeit there are no studies available that describe the Ag-specific T cell response to phages, the sequential induction of IgM and IgG upon immunization provides evidence that T cells specific for phage Ag develop and enable class switch recombination (45). In theory, this bears the potential that adaptive immune responses could be avoided by preselection of phages with low immunostimulatory potential.

In a murine model of bacteremia with antibiotic resistant E. coli and P. aeruginosa strains, an increase in anti-phage IgG levels (58, 59) was detectable on day 10 after administration of 109 PFU of the respective phages but had no effect on survival because of the short disease course, which only lasted for a few days. IgG levels reached the maximum ∼20-fold increase after 30 and 40 d, respectively. In an earlier report on acute bacteremia, the authors showed that despite using similar doses (1010 PFU), anti-phage IgM and IgG only became detectable after three repetitive phage injections and levels (5-fold and 3800-fold over baseline, respectively). The levels remained unchanged despite further phage injections (60).

An early study in immune-deficient mice highlighted a role for B cells in clearance of phages after i.v. injection (61). Although clearance occurred within an hour in wild type mice, phage titers persisted in both SCID and B cell–deficient mice. More recent in vivo studies demonstrated that anti-phage Abs mediate clearance of phages from the gut and circulation: increasing levels of phage-specific IgA in feces correlated with gradual absence of orally ingested phages (62) and high levels of anti-phage IgG, with elimination of phages from the blood and tissues after i.p. administration (63).

Furthermore, kinetic studies of phage circulation in the peripheral blood of healthy and X-linked agammaglobulinemia patients provided evidence that elimination of phages in humans may similarly require opsonization by Abs. Whereas in normal subjects injected with ϕX174, phages were completely eliminated within 4 d, in patients with very low Ab titers, phages remained detectable in the circulation for up to 7 wk (64).

Neutralizing Abs constitute a risk for therapeutic failure in phage therapy. Notably, early electron microscopy studies using the E. coli–specific ssDNA phage M13 associate Ab-mediated neutralization with mechanical hindrance of bacterial cell penetration due to bound Ig (65).

Two studies on patients receiving phage therapy are available. In a study from 1987, anti-phage Ab titers were studied in 57 patients before and after phage therapy for different types of infections (66). In 13 cases, anti-phage Abs were present before initiation of therapy and increased upon therapy; in 17 cases, anti-phage Abs became detectable under therapy. The authors emphasize that only in two cases did the Ab titers before therapy exceed a 1:80 dilution of the serum. In these two cases, the outcome of therapy was unsatisfactory. For all others, the presence and induction of anti-phage Abs did not lead to therapy failure. However, because of the low number of cases and in the absence of a rigorous statistical analysis, the results remain descriptive. The data collected in a more recent study on 20 patients with Staphylococcus aureus infections reveal that there is significant induction of anti-phage IgM and, in particular, IgG titers caused by therapy (67). Neutralizing activity in sera increases with the Ab titers. In addition, generation of IgG is strongly dependent on the individual phage. The data do not allow conclusions on the predictive value of neutralizing Ab titers in regard to patient outcome.

Taken together, these studies indicate that the presence of phage-specific Abs can interfere with therapeutic efficacy. This is particularly relevant in chronic infections in which repeated treatments with the same phages boost the humoral immune response. Conversely, the induction of phage-specific Ig seems irrelevant for treatment of acute infections because antibacterial effects of phages become effective before Abs are formed. However, prescreening of patient sera for the absence of anti-phage Abs could be advisable before initiation of therapy. The delay may, however, not be compatible with acute treatment. A possible solution for pre-existent anti-phage Abs is presented in a recent report that proposes that packaging of phages into liposomes not only promotes cellular uptake into infected macrophages but avoids binding of anti-phage Abs and neutralization (68). Nevertheless, this approach might not be feasible in infections with extracellular bacteria. Here, genetic deletion of phage genes encoding immunogenic proteins might represent an interesting alternative for sustenance or rescue of phage efficacy. This approach would, however, require an extensive characterization of phages with the scope of comprehensive identification of potentially immunogenic phage proteins, which, considering the currently applied methods, is difficult to achieve. Ultimately, genetically engineered lytic phages would be applied to patients, which could raise additional regulatory concerns.

Taken together, the vast majority of data indicate that therapeutic application of high-titer bacteriophages exposes them to and stimulates the host immune system. Although some studies describe dose- and time-dependent immunological effects, few were conducted with phages used for antibacterial therapy. Moreover, the relevancy of the systemic anti-phage immune response has to be assessed in conjunction with duration of treatment until recovery and route of administration. This has, so far, not been systematically addressed in patients or animal models.

Three major areas of phage-immune interaction can be discerned:

First, immune recognition via PRR mainly contributes to resolution of infection by recruiting phagocytes to the infection site (11). Phage-mediated activation of innate immune cells is mainly based on recognition of phage-derived DNA and RNA by PRR. Specific PRR engagement and the extent of immune activation, therefore, will differ depending on the phage type, the phage dose, and nucleic acid synthesis activity. Not surprisingly, successful treatment of infections with multidrug-resistant pathogens was primarily dependent on phage specificity and susceptibility of the infecting bacterial strains (59, 60, 69). However, although phage therapy may be fully effective in lymphopenic patients, it might be impaired in patients with myeloid cell deficiencies.

Second, immunogenicity of phages promotes the formation of phage-neutralizing Abs that can hamper therapeutic success and increase with repeated administration (60). Although nucleic acid–sensing PRR drive the process of Ab generation (46), the arising Abs are directed at immunogenic proteins present on the phages (29, 70). At present, it is not known whether selection of natural phages from different sources is biased toward more or toward less immunogenic phages. Protein expression is considered a characteristic of the individual phage; Ab induction is, therefore, predicted to be highly variable, and immunogenicity should be taken into account in the selection of phages for therapeutic purposes. Additionally, and despite its challenges, genetic engineering of phages to abrogate the expression of immunogenic proteins might represent an interesting approach to avoid therapeutic inefficacy due to the presence of phage-specific Ig in repeated administration courses of well-characterized immunogenic phages. For the time being, avoidance of PRR-mediated immune effects could lead to reduction of phage-induced neutralizing Ab formation. This could be achieved by dose reductions based on the use of self-amplifying phages or refined dose-ranging studies.

Third, phage-specific IgG or IgA can limit the multiplication rate of phages. High Ab levels and Fc receptor–mediated uptake of phage/Ab complexes by macrophages promote the clearance of phages from the human body (62, 63). However, to date, it is not well understood whether this regulatory role of anti-phage Abs could also be important for prevention of resistance development to phages and, additionally, whether pre-existing immunity to natural phages could affect phage therapy. Furthermore, it is unclear which phage-specific factors have impact on this specific mechanism of clearance. Thus, more in-depth investigations are warranted to elucidate whether phage persistence due to lack of immune control (64) might favor selection of phage-resistant bacterial strains in vivo.

Together, the compiled data indicate that there are gaps in our understanding of the clinical relevancy of the phage-immune interaction. Nevertheless, immunogenicity of phages itself does not seem to represent a relevant safety risk for patients. However, many studies refer to phages selected for diagnostic immunization or tumor therapy. The results obtained in these studies cannot be extrapolated to lytic phages, and reports on the immune effects of clinically applied lytic phages are very limited. Furthermore, validated assays for standardized measurement of in vitro and in vivo immune effects exerted by therapeutic candidate phages are outstanding despite the importance of these findings from a clinical and regulatory perspective.

At present, the clinical value of phage therapy remains uncertain. This literature review reveals that, more recently, reports are increasingly addressing the major gaps in our understanding of the immunological aspects of phage therapy, mainly in in vivo infection models. However, despite a continuously increasing number of publications on phage therapy, there are few studies using validated methods and rigorous controls. The introduction of validated in vitro and in vivo methods is indispensable, as it will permit the assessment of comparability of immune effects of different phages and phage combinations. Only these validated methods will allow valid conclusions on the key issues, such as 1) the value of immune-based parameters for selection of phages and identification of responsive patient populations and 2) exchangeability of phages, an important prerequisite for individualized phage therapy concepts.

Abbreviations used in this article:

     
  • PAMP

    pathogen-associated molecular pattern

  •  
  • PRR

    pattern recognition receptor.

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The authors have no financial conflicts of interest.