Bacteriophages are promising antibacterial agents. Although they have been recognized as bacterial viruses and are considered to be non-interacting with eukaryotic cells, there is growing evidence that phages may have a significant impact on the immune system via interactions with macrophages, neutrophils, and T-cell polarization. In this study, the influence of phages of podovirus, siphovirus, and myovirus morphotypes on humoral immunity of CD-1 mice was investigated. In addition, tissue distribution of the phages was tested in these mice. No common patterns were found either in the distribution of phages in mice or in changes in the levels of cytokines in the sera of mice once injected with phages. Importantly, pre-existing IgM-class antibodies directed against capsid proteins of phages with myovirus and siphovirus morphotypes were identified in mice before immunization. After triple immunization of CD1-mice with phages without any adjuvant, levels of anti-phage serum polyclonal IgG antibodies increased. Immunogenic phage proteins recognized by IgM and/or IgG antibodies were identified using Western blot analysis and mass spectrometry. In addition, mice serum collected after immunization demonstrated neutralizing properties, leading to a substantial decrease in infectivity of investigated phages with myovirus and siphovirus morphotypes. Moreover, serum samples collected before administration of these phages exhibited some ability to reduce the phage infectivity. Furthermore, Proteus phage PM16 with podovirus morphotype did not elicit IgM or IgG antibodies in immunized mice, and no neutralizing activities against PM16 were revealed in mouse serum samples before and after immunization.

Ulcerative colitis is a chronic immune-mediated disease of unclear etiology, affecting people of different ages and significantly reducing the quality of life. Modern methods of therapy are mainly represented by anti-inflammatory drugs and are not aimed at a specific pathogenetic factor. In this study, we investigated the effect of transplantation of sterile stool filtrate from healthy donors on the induction of anti-inflammatory immune mechanisms. It was shown that performing such a procedure in patients with ulcerative colitis caused the appearance of T helper cells in the blood, which reacted to the content of sterile stool filtrates in an antigen-specific manner and produced IL-10. At the same time, cells of the same patients before therapy in response to the addition of sterile stool filtrates were less reactive and predominantly produced IL-4, indicating its pro-inflammatory skewing. The obtained data demonstrated the effect of an anti-inflammatory shift in the T-helper response after transplantation of sterile stool filtrate, which increased and persisted for at least three months after the procedure.

Bacteriophages, traditionally viewed solely as antibacterial agents, are increasingly being studied for their immunomodulatory properties. In this study, we demonstrate that PM16 phage therapy not only effectively controls subcutaneous Proteus mirabilis infection in mice but also induces long-term specific humoral immunity against subsequent reinfection. This immunomodulatory effect was dose-dependent. In vitro, PM16 directly activates macrophages, leading to increased production of proinflammatory cytokines (tumor necrosis factor-α and interleukin-1β) and inducible nitric oxide synthase, and enhances macrophage bactericidal activity against P. mirabilis. We assume that the enhancement of the adaptive immune response is mediated not by the phage acting as a classical antigenic adjuvant but by its ability to prime innate immune cells, specifically macrophages. This priming leads to more efficient bacterial clearance, antigen presentation, and the formation of protective immunological memory.

Bacteriophages (phages) represent promising therapeutic agents. Their clinical use is challenged by the rapid rise of resistant bacterial clones. To overcome this problem, phages can be trained in vitro to improve their ability to cope with the possible resistance that may arise. Here, we co-evolved phages with their hosts under different conditions and assessed their ability to infect an adapted bacterial panel using qPCR. The co-evolution experiment yielded a panel of bacterial clones adapted either to a phage, a competing phage, or a cocktail of both. Phages were adapted either in the continuous presence of an evolutionarily naïve host, in a cocktail with a competing phage, under both conditions, or under neither condition. We assessed each resulting phage for its ability to infect evolved bacterial clones in the panel we created, using qPCR for preliminary high-throughput assessment. This allowed us to evaluate 500 phages–bacteria interactions. Overall, qPCR-detected phage production following infection of different bacterial clones improved to varying degrees when evolutionary naïve hosts were added during the training. However, the screening suggests that optimal training conditions are phage-specific. For Enterobacter cloacae phages EC151 and EC152, the broadest increase in qPCR-estimated phage production in our experiments was observed when a competing phage and/or an evolutionarily naïve host was included during adaptation. For Stenotrophomonas maltophilia phages StM171 and StenM174, the presence of an evolutionarily naïve hosts appeared beneficial in both replicates; co-adaptation with a competing phage led to a complete loss of StM171 infectivity in both experiments but benefited StenM174. Phages passaged for ten passages consistently infected a broader range of bacterial clones than those sampled after five passages. Sequencing of eight EC152-derived phages identified recurring mutations in a transcriptional regulator, and in some cases, in baseplate and tail fiber genes.

Bacteriophages (phages) represent promising therapeutic agents. Their use in treatments is challenged by the rapid rise of resistant bacterial clones. To overcome this problem, phages can be trained in vitro to adapt them to the possible resistance that may arise. Here, we co-evolved phages with their hosts under different conditions and assessed the outcomes using qPCR. The co-evolution experiment yielded a panel of bacterial clones that were either adapted to a phage, a competing phage, or to a cocktail of both. The adaptation of a phage was done either in the continuous presence of an evolutionarily naïve host, or in a cocktail with a competing phage, or both conditions, or neither conditions. We assessed each obtained phage ability to infect evolved bacterial clones in the panel we created, and we used qPCR to enable high-throughput assessment. This allowed us to evaluate 500 phage-bacteria interactions. While all phages benefitted from the presence of evolutionary naïve hosts, the screening suggests that optimal training conditions are phage-specific, based on the four phages tested. For Enterobacter cloacae phages EC151 and EC152, the most extensive infectivity in our experiments was observed when a competing phage and/or an evolutionarily naïve host was included during adaptation. For Stenotrophomonas maltophilia phages StM171 and StenM174, the presence of an evolutionarily naïve hosts appeared beneficial in both replicates; co-adaptation with a competing phage led to a complete loss of StM171 infectivity in both experiments, but benefited StenM174. Phages passaged for 10 passages consistently infected a broader range of bacterial clones than those sampled after 5 passages. Sequencing of 8 phages obtained after adapting EC152 identified recurring mutations in a transcriptional regulator, and in some cases, in the baseplate and tail fiber genes.

Considering the therapeutic potential of the Proteus mirabilis PM16 podophage, the interaction between PM16, its host strain, and the mouse immune system was investigated. We evaluated how pre-existing humoral immunity to PM16 influences the immune response against P. mirabilis and the neutralization of the phage itself. Balb/c mice were divided into three groups and immunized two times with (1) 0.9% NaCl, (2) adjuvants, or (3) a mixture of PM16 and an adjuvant. Then, each group was subdivided into three subgroups: mock infection, infection with P. mirabilis, and infection with P. mirabilis followed by model phage therapy with PM16. The obtained results demonstrated that pre-immunization with PM16 enhanced the anti-P. mirabilis IgG antibody response upon bacterial challenge, indicating that the phage potentiates antibacterial immunity. In addition, pre-immunization elicited a significant anti-PM16 antibody response that resulted in in vitro neutralization of phage lytic activity. However, phage-neutralizing antibodies neither decreased the efficacy of phage therapy nor influenced bacteria-specific immune response. Thus, while PM16 can boost the host’s immune response against its bacterial host, the resulting humoral immunity also drives phage clearance through both direct and bacteria-mediated neutralization pathways, revealing a complex immunopharmacological relationship central to phage therapy.