Phage therapy represents a promising strategy to tackle the growing threat of antimicrobial resistance. Increasing evidence has demonstrated that phage-specific antibodies may compromise the efficacy of phage therapy. Nevertheless, little is known about how phage protein specificity of antibodies modulates phage therapeutic outcomes. Herein, we utilized AbP20, a podovirus against Acinetobacter baumannii, to explore the effects of antibodies elicited by each structural protein on phage therapy efficacy in a mouse infection model. Intraperitoneal administration of AbP20 once a day for 7 consecutive days induced robust phage-specific antibody responses that impaired phage therapy. Genome-guided antigen screening identified that antibodies elicited by the nozzle and fiber proteins of AbP20, rather than those targeting the portal, capsid, or adaptor proteins, are the dominant drivers of phage therapy failure. Specifically, fiber-specific and nozzle-specific antibodies block bacterial adsorption and genomic injection of AbP20, respectively. Meanwhile, both antibody subsets efficiently induce large phage aggregates and potentiate macrophage phagocytosis via an Fc receptor-independent pathway. An evolved AbP20 variant with improved neutralization escape capacity exhibited comparable antibody-accelerated phagocytosis and host immune clearance, yet partially rescued therapeutic failure caused by neutralizing antibodies. Collectively, this study elucidates that nozzle- and fiber-elicited neutralizing antibodies impair phage therapy via dual synergistic mechanisms, which offers novel insights into the multifaceted modulation of phage-specific antibodies against phage therapeutic efficacy.

AbP20-induced antibodies impair phage therapy

Phage AbP20 is a lytic podovirus isolated from hospital wastewater in Wuhan, with A. baumannii strain Ab28 serving as the propagation host. It presents typical T7-like morphology consisting of an icosahedral capsid and a short tail (Fig 1A). Both bacterial growth inhibition and dynamic bactericidal assays verified that AbP20 exhibits potent bactericidal activity against Ab28 across different MOI gradients (Figs 1B and S1A). Whole-genome sequencing analysis revealed that AbP20 harbors a 42.023 kb double-stranded DNA genome (GSA accession: CRA031834), which encodes a total of 49 predicted open reading frames (S1B Fig).

Given the potent bactericidal activity of phage AbP20, we used it as a model to evaluate the effects of phage-specific antibodies on therapeutic outcomes. We first evaluated the therapeutic efficacy of phage AbP20 in a mouse model of Ab28 infection (Fig 1C). To this end, BALB/c mice were intraperitoneally injected with 2 × 10⁸ CFU/mouse of Ab28 and then received an intraperitoneal administration of phage AbP20 at an MOI of 1 or 10 at 1 and 8 h post‑infection. Results showed that all mice in the PBS control group succumbed to death within 24 h post infection (Fig 1D). In contrast, phage-treated animals exhibited a 100% survival rate, comparable to the antibiotic-treated group (n = 5, Fig 1D). Specifically, 24 h post-infection, bacterial loads in the liver, spleen, and kidneys were significantly decreased in both the antibiotic-treated group and the phage-treated group (n = 5, Fig 1E). Notably, at 10 h post-infection, the high dose AbP20-treated group (MOI = 10, 2 × 10⁹ PFU/mouse) exhibited a balanced hematopoietic cell profile, with proportions of lymphocytes, neutrophils, monocytes, platelets and leukocytes comparable to the uninfected mock group (Figs 1F, 1G and S2), indicating that AbP20 phage at MOI = 10 effectively promotes bacterial clearance and protects mice from lethal A. baumannii infection. Moreover, AbP20 exhibited a favorable safety profile after repeated intraperitoneal administration in mice, demonstrating minimal impact on body weight gain, peripheral blood immune cell counts, and organ histopathology (S3 Fig).

Next, we asked whether repeated exposure to AbP20 triggers immune responses that render phage therapy ineffective. To address this issue, mice were administrated 2 × 10⁹ PFU/mouse of AbP20 intraperitoneally once a day for 7 consecutive days and then maintained for another 4 weeks to allow phage-specific antibody generation. Afterwards, mice were infected intraperitoneally with a lethal dose of A. baumannii followed by two doses of AbP20 (Fig 1H). Mice were tested for phage-specific antibody levels 10 days before infection. As expected, the serum collected from the phage-stimulated group contained high level phage-specific antibody (Fig 1I), which reduced the efficiency of plating (EOP) of phage AbP20 by four orders of magnitude (Fig 1J), indicating strong neutralization activity. In the subsequent infection experiment, administration of 2 × 10⁹ PFU/mouse of AbP20 only showed 33.3% protection (Fig 1K) and limited bacterial clearance efficacy (Fig 1L) in the phage-stimulated group (n = 6). These results collectively indicate that phage-induced specific immune responses hinder bacterial clearance in vivo.

Fiber- and nozzle-induced antibodies lead to the failure of phage therapy

Genome-based analysis showed that phage AbP20 contains five structural proteins, i.e., the capsid protein (gp37), the portal protein (gp35), the adaptor protein (gp39), the nozzle protein (gp40), and the fiber protein (gp44) (Fig 2A). To identify which phage protein-elicited antibodies are capable of impairing phage therapy, we expressed genes coding for the five structural proteins using an E. coli expression system (S4A Fig). Afterwards, BALB/c mice were intramuscularly immunized with each structural protein to induce sufficient protein-specific antibodies, and the therapeutic efficiency of AbP20 was evaluated in a mouse model of A. baumannii infection (Fig 2B). Similar to the AbP20-challenged group, each individual phage protein elicited strong humoral immune responses as determined 3 days before A. baumannii infection (S4B Fig) with minor differences among varied IgG types (S4C–S4F Fig). Strong serological cross-reactivity was observed between sera from mice challenged with each structural protein and whole phage particle, indicating that the proteins produced from the expression system were antigenically relevant to native proteins present in the phage virion (Fig 2C). Concordantly, immunization with phage AbP20 also induced antibodies reactive against each structural protein (Fig 2D). Notably, the proteins gp40 and gp44 exhibit high cross-reactivity with the intact phage AbP20, although the capsid protein (gp37) is the most abundant component within the phage virion, suggesting differences in immunogenicity among phage structural components.

In the subsequent challenge assay, the same as the phage-stimulated group, immunization with either gp40 or gp44 led to therapeutic failure, whilst immunization with gp37, gp35, and gp39 did not affect the efficacy of phage therapy (n = 6, Fig 2E). In line with these observations, phage AbP20 failed to remove A. baumannii from organs in groups stimulated with either AbP20, gp40, or gp44 as did in groups stimulated with gp35, gp37, and gp39 (n = 4, Fig 2F). Further analysis showed that, although slightly weaker than those induced by the intact phage particles, sera from gp40- and gp44-immunized groups, but not from the gp35-, gp37-, and gp39-immunized groups, exhibited strong phage neutralization capabilities and markedly suppressed phage proliferation (Figs 2G and S5A, S5B). Specifically, fiber protein gp44-elicited antibodies blocked the adsorption of phage AbP20 onto host bacterial cells (Figs 2H and S5C), whereas gp40-specific antibodies selectively inhibited phage genomic injection without interfering with bacterial adsorption (Fig 2I). Such two distinct inhibitory modes ultimately caused a sharp decline in progeny phage virion release (Fig 2J). Altogether, antibodies induced by the fiber and nozzle proteins of AbP20 are probably the major antigenic components of the phage virion that are responsible for inducing antibodies impairing therapeutic outcome.

The data underlying this figure can be found in S1 Data.

Fiber- and nozzle-induced antibodies promote the clearance of AbP20 by facilitating phagocytosis

It is anticipated that phage-specific antibodies may promote the immune clearance of phages in vivo through multiple mechanisms, one of these being antibody-mediated opsonophagocytosis. Therefore, we used murine macrophage RAW264.7 cells to examine the influence of each structural protein-induced antibody on the phagocytosis of AbP20. In this regard, phages (with a final endotoxin level of 76.23 EU/ml to minimize their effects on macrophage cells) were preincubated with serum from the mice immunized with either a single structural protein or the entire AbP20 at 37 °C for 1 h, after which the mixture was cocultured with macrophage cells for phagocytosis analysis (Fig 3A). Phage engulfment by the cells was assessed by immunodetection of the phage capsid protein (gp37) using rabbit anti-gp37 antibodies. Compared to the PBS-treated group, the mass of gp37 within cells was significantly increased in the presence of AbP20-specific serum up to dilution factor of 320 folds (Fig 3B, 3C), suggesting that AbP20-immunized serum promotes the phagocytosis of AbP20 by macrophages. This conclusion was further supported by confocal fluorescence microscopy (S6 Fig). We then examined whether the antibodies induced by individual structural proteins could promote phage uptake. To our surprise, antibodies induced by the nozzle protein (gp40) and the fiber protein (gp44) significantly enhanced the phagocytosis of AbP20 by macrophage (Fig 3D, 3E). In contrast, antibodies induced by other structural proteins, including the portal protein (gp35), gp37, and the adaptor protein (gp39), did not exhibit significant effects on phage phagocytosis (Fig 3D, 3E). Confocal microscopy further illustrated that gp40 and gp44 immune sera markedly enhanced the internalization of AbP20 by macrophage (Fig 3F), with significantly increased intracellular fluorescence intensity (Fig 3G), whilst differences were not observed in groups treated with gp35-, gp37-, and gp39-specific serum as compared to the native serum (Fig 3F, 3G).

To further validate this assumption, we assessed the clearance of AbP20 in BLAB/c mouse positive to antibodies elicited by each single structural protein, or the entire phage AbP20 (n = 3, Fig 3H). Results showed that significantly reduced phage titers were detected in spleens from groups immunized with gp40, gp44, or entire phage AbP20 in all time points detected as compared to groups immunized with gp35, gp37, and gp39 (Fig 3I). Altogether, these findings demonstrate that antibodies elicited by the nozzle and fiber proteins of AbP20 destroy phage activity via dual mechanisms: suppressing phage propagation and potentially promoting immune clearance, thereby impairing phage therapy. By contrast, antibodies elicited by the remaining structural proteins of AbP20 exert negligible impacts on the outcome of phage therapy.

Antibody‑induced phage agglutination modulates phage phagocytosis

Since the phagocytotic efficiency is governed by the size and shape of cargoes [36], the fact that AbP20-elicited antibodies promote the phagocytosis of AbP20 by macrophage led us to think whether phage-specific antibodies induce the agglutination of phages and thereby affecting their phagocytosis. To investigate this, we first determined the size of phage in the presence of varied immune serum. As expected, enlarged sizes of AbP20 aggregates were observed in the presence of serum immunized with AbP20, the nozzle protein (gp40), and the fiber protein (gp44), but not in the serum immunized with the portal protein (gp35) and the adaptor protein (gp39), as compared to the PBS-treated group (Fig 4A), suggesting that gp40- and gp44-elicited antibodies induced the agglutination of AbP20 and thus facilitating its phagocytosis.

To make it clear, we purified IgG with high titers from different immune sera using protein G resin (S7A Fig) and found that, resembling the immune serum, purified IgG against gp40, gp44, and the whole phage particle exhibited strong neutralizing activity and promoted phage phagocytosis by macrophages (S7B, S7C Fig). We then detected the status of AbP20 aggregates in the presence of varied IgG using transmission electron microscopy. As shown in Fig 4B, varied sizes of aggregates were indeed observed in the presence of IgG obtained from AbP20-, gp40-, gp44-, and gp37-immunized serum, whilst aggregates were not observed in the presence of IgG obtained from gp35-, gp39-, and the PBS-immunized serum (Vehicle). Detailed observation of antibody-mediated phage aggregates revealed that the aggregation pattern induced by antibodies targeting gp37 differs from those induced by antibodies targeting gp40 and gp44 (S8 Fig), since in principle, head-targeting antibodies may trigger phage assembly into structures featuring head-to-head juxtaposition, with the spike-like phage tails exposed outward. In contrast, antibodies targeting tail components may induce tail-to-tail phage clustering, where phage spherical capsids oriented toward the exterior.

Size of antibody-induced phage aggregates regulates phagocytosis in an Fc receptor-independent manner

It is well established that Fc receptor engagement facilitates phagocytosis and immune clearance via multiple effector pathways, including antibody‑dependent cellular phagocytosis [37]. Antibodies elicited by the capsid protein (gp37) exhibited a comparable serotype profile (S4 Fig) but exerted disparate effects on macrophage-mediated phagocytosis (Fig 3E), which prompted us to explore the role of Fc receptor-mediated phagocytosis in this process. To this end, we examined the effects of structural protein-specific immune serum on RAW264.7 cell-mediated phagocytosis of AbP20 upon Fc receptor blockade. Intriguingly, no obvious disparities in antibody-triggered phagocytosis were detected between the Fc receptor-blocked groups (FcR-block) and isotype control groups (Fig 5A), revealing that the nozzle protein (gp40)- and fiber protein (gp44)-specific antibodies accelerate AbP20 phagocytosis in an Fc receptor-independent manner.

We next investigated whether antibody-potentiated phagocytosis is solely driven by the formation of large phage aggregates. For this purpose, we purified Fab fragments targeting gp37, gp40, gp44, and intact AbP20 via papain digestion of corresponding purified IgGs (Fig 5B). After verifying the neutralizing activity of all prepared Fab fragments (Fig 5C), we assessed their impacts on phage agglutination and subsequent macrophage phagocytosis. Results showed that phage aggregates formed in the Fab-treated groups were drastically smaller than those formed in the full-length IgG-treated groups (Fig 5D). Consistently, the enhanced RAW264.7 phagocytosis of AbP20 induced by gp40-specific, gp44-specific and AbP20-specific IgGs was abrogated after treatment with their matching Fab fragments (Fig 5E–5H). Taken together, these findings indicate that antibody-mediated formation of large phage aggregates acts as the predominant driver of enhanced macrophage phagocytosis of therapeutic phages, wherein the IgG Fc domain plays an indispensable regulatory role that requires further mechanistic exploration.

Evolved neutralization escape partially ameliorates antibody-mediated impairment of phage therapy

All the above observations indicate that antibodies elicited by the nozzle protein gp40 and the fiber protein gp44 impair phage therapy via potent neutralization and enhanced phagocytosis of AbP20. As a means to delineate the contribution of each effector mechanism, we conducted in vitro phage evolution by incubating wild-type AbP20 with serially diluted AbP20-immune serum for a total of 10 passages (Fig 6A). Consequently, we obtained a phage variant harboring mutation in the nozzle protein gp40 (Y305H and T656G) and the fiber protein gp44 (Q131K), designated AbP20M (Fig 6B). This mutant exhibited enhanced neutralization escape capability (Fig 6C) and retained comparable bacteriostatic activity relative to wild-type AbP20 (S9A Fig). We further evaluated the responsiveness of AbP20M to antibody-mediated agglutination and macrophage phagocytosis. Results demonstrated that IgGs purified from serum against the capsid protein (gp37-IgG), nozzle protein (gp40-IgG), fiber protein (gp44-IgG), and intact AbP20 (AbP20-IgG) triggered AbP20M aggregation with morphological features and aggregate sizes comparable to those of wild-type AbP20 (Fig 6D, 6E). Consistent with these results, RAW264.7 macrophage cells displayed nearly identical phagocytic efficiency toward free AbP20M and wild-type AbP20 (S9B Fig). Resembling wild-type AbP20, AbP20M phagocytosis by RAW264.7 cells was also markedly promoted by gp40-IgG, gp44-IgG and AbP20-IgG (Fig 6F–6H). Next, we assessed the in vivo clearance rate of AbP20M in mice immunized against each individual structural protein or intact AbP20 (n = 3). Results revealed that the time-dependent splenic clearance of AbP20M in mice immunized with gp40, gp44 or phage AbP20 was markedly lower than that in the other groups (S9C Fig), which was consistent with the clearance pattern of wild-type phage AbP20. These observations collectively suggest that the evolved variant AbP20M confers enhanced neutralization escape capacity while possessing equivalent immune clearance properties relative to wild-type AbP20.

Furthermore, we evaluated the therapeutic efficacy of AbP20M in an A. baumannii-infected mouse model pre-immunized with intact AbP20 prior to phage therapy (n = 6, Fig 6I). As expected, intramuscular immunization elicited high antibody titers against AbP20 (Fig 6J), which exerted potent neutralizing activity (Fig 6K). Accordingly, negligible protection was observed in mice treated with wild-type AbP20 at doses of 10 or 100 MOI (Fig 6L), indicating that AbP20-elicited antibodies thoroughly impair phage therapeutic effects. In contrast, dose-dependent protective effects were observed in AbP20M-treated groups. Specifically, one of six mice survived in the 10 MOI AbP20M treatment group (Fig 6L), while the survival rate rose to 50% at an AbP20M dose of 100 MOI (Fig 6L), accompanied by markedly reduced tissue bacterial burden (Fig 6M). Collectively, these results demonstrate that the evolutionary acquisition of resistance to phage-elicited antibody neutralization can partially improve phage therapy efficacy; nevertheless, antibody-mediated phagocytosis and immune clearance remain major barriers that cannot be fully overcome.

Based on all these observations, we propose a working mechanistic scenario for antibody-mediated phage therapy outcomes. Antibodies targeting distinct structural proteins of phage AbP20 exert disparate regulatory effects on phage fate and therapeutic performance. Specifically, antibodies against the capsid protein (gp37), portal protein (gp35), and adaptor protein (gp39) mainly serve as binding antibodies, which exert negligible impacts on phage infectivity and macrophage-mediated phagocytosis, and barely alter final phage therapeutic efficacy. By comparison, antibodies induced by the fiber protein (gp44) and nozzle protein (gp40) act as dominant neutralizing antibodies. These two types of antibodies suppress phage proliferation via blocking phage-host interaction and accelerate systemic phage clearance by triggering the formation of large phage aggregates, thereby compromise the therapeutic potency of phage AbP20 (Fig 7).

Ethics statement

All mouse experiments were conducted in an ABSL-2 lab, and all experimental methods were carried out in accordance with the regulations and guidelines set forth by the Animal Experiments Committee of the Wuhan Institute of Virology, Chinese Academy of Sciences. All experimental protocols were approved by the Animal Experiments Committee of Wuhan Institute of Virology, Chinese Academy of Sciences (WIVA41202201).

Phage purification

Phage AbP20 was added at an MOI of 0.01 to the exponentially growing culture of A. baumannii strain Ab28 and cocultured for 4–6 h to allow phage proliferation. Afterwards, supernatant of the phage culture was harvested by centrifugation for 10 min at 8,000 rpm, then 1 M NaCl and 10% PEG 8,000 were added to the supernatant and stored at 4°C overnight for phage precipitation. Precipitated phage pellet collected by centrifugation at 10,000 × g for 1 h was resuspended in PBS and mixed with an equal volume of chloroform. Following centrifugation at 3,500 rpm, phage was collected from the upper aqueous phase and subjected to CsCl density gradient centrifugation at 180,000 × g for 2 h to remove lipopolysaccharide and bacterial contaminants. Phage preparations were filtered through a 0.22 μm filter and stored at 4 °C after extensive dialysis. Endotoxin concentration was determined by the endpoint chromogenic method using the ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript Biotech Corp., USA) according to manufacturer’s instructions. Phage titer was determined by the plaque assay described below.

Phage plaque assay

Phage quantification was performed using the double-agar overlay plaque assay. Briefly, A. baumannii Ab28 was overlaid onto an LB (lysogeny broth) solid agar base in 0.7% soft agar. Serially diluted phage AbP20 was then spotted onto the double-layer plates. After incubating at 37 °C for 6 h, plaques were counted and phage titer was calculated.

Negative-stain transmission electron microscopy

A phage suspension at a concentration of 10¹¹ PFU/ml was applied to a carbon-coated copper grid for 1 min, followed by negative staining with 2.0% phosphotungstic acid (pH 7.0) for 2 min. Electron images were recorded on a Talos L120C transmission electron microscope.

Bactericidal activity of AbP20 against A. baumannii Ab28

To test the inhibitory effects of AbP20 on the growth of A. baumannii, exponential cultures of Ab28 were diluted with LB to an OD₆₀₀ of 0.4 and incubated with different MOIs (0, 0.01, 0.1, 1, and 10) of AbP20 at 37 °C with shaking at 180 rpm. The turbidity of bacterial culture at 600 nm was monitored every 30 min for 12 h. To determine the bactericidal activity of phage AbP20 against Ab28, log‑phase culture of Ab28 was cultured in LB at 37 °C with shaking at 200 rpm in the presence of AbP20 an MOI of 1. Viable bacteria were quantified by enumerating the CFU at 0, 1, 2, 4, and 6 h of coculture. All experiments were carried out in triplicate.

Purification of phage structural proteins

Genes coding for each individual structural protein of AbP20, i.e., gp35, gp37, gp39, gp40, and gp44, were cloned into the pET-28a(+) expression vector with a C‑terminal 6 × His tag using gene-specific primers (S1 Table) and transformed into E. coli BL21(DE3) for expression. Recombinant proteins were purified through Ni‑NTA affinity chromatography and dialyzed against PBS. Protein concentration and purity were assessed using NanoDrop spectrophotometry and SDS‑PAGE.

Mouse experiments

To assess the safety of AbP20, 6–8-week-old female BALB/c mice were intraperitoneally administrated with 100 μL of 1 × 10¹⁰ PFU/mL AbP20 daily for 7 consecutive days. Body weights were monitored daily for 10 days. One day after the last phage administration, peripheral blood from each mouse was collected for hematological analysis, the heart, liver, spleen, lungs, kidneys, and brain were harvested for histopathological examination.

To evaluate the efficacy of phage therapy in the absence of phage-specific antibodies, 6–8-week-old female BALB/c mice were infected intraperitoneally with 200 μL of 1 × 10⁹ CFU/mL A. baumannii Ab28. At 1- and 8-h post-infection, mice were intraperitoneally administrated with either 30 mg/kg apramycin, 2 × 10⁸ PFU/mouse phage AbP20, 2 × 10⁹ PFU/mouse AbP20, or an equal volume of PBS buffer. Survival in each group was monitored every 12 h for 6 days (n = 5). At 10 h post infection, peripheral blood was collected from each group for hematological analysis (n = 3). At 24 h post infection, the liver, kidney, and spleen were harvested from each group for CFU enumeration (n = 5).

To evaluate the efficacy of phage therapy after repeated exposure to AbP20, 6–8-week-old female BALB/c mice were intraperitoneally administrated with either 100 μL of AbP20 (1 × 10¹⁰ PFU/mL) or an equal volume of PBS once daily for 7 consecutive days. Four weeks after the last stimulation, mice were intraperitoneally challenged with 2 × 10⁸ CFU/mouse of A. baumannii Ab28 and then received 2 × 10⁹ PFU/mouse of AbP20 intraperitoneally at 1- and 8-h post‑infection. Survival was monitored every 12 h for 6 days (n = 6). At 24 h post infection, the liver, kidney, and spleen were harvested from each group for CFU enumeration (n = 6). Ten days before infection, serum from each group was collected via retro‑orbital bleeding for antibody titer determination.

To evaluate the efficacy of phage therapy in the presence of phage-specific antibodies, 6–8-week-old female BALB/c mice were immunized intramuscularly with either 5 × 10¹⁰ PFU/mouse of AbP20, 10 μg aluminum adjuvanted each of gp35, gp37, gp39, gp40, and gp44, or an equal volume of PBS with aluminum adjuvant three times at 2-week intervals. Two weeks after the last boost, mice were intraperitoneally challenged with 2 × 10⁸ CFU/mouse of A. baumannii Ab28 and then received 2 × 10⁹ PFU/mouse of AbP20 intraperitoneally at 1- and 8-h post‑infection. Survival was monitored every 12 h for 6 days (n = 6). At 24 h post infection, the liver, kidney, and spleen were harvested from each group for CFU enumeration (n = 4). Three days before infection, serum from each group was collected via retro‑orbital bleeding for antibody titer determination.

To determine phage clearance in spleen with the presence of phage-specific antibodies, 6–8-week-old female BALB/c mice were intramuscularly immunized with either 5 × 10¹⁰ PFU/mouse of AbP20, 10 μg aluminum adjuvanted each of gp35, gp37, gp39, gp40, and gp44, or an equal volume of PBS with aluminum adjuvant three times at 2-week intervals (n = 3). Two weeks after the last boost, mice were intraperitoneally administered with 5 × 10⁹ PFU/mouse AbP20 or AbP20M. At 1-, 3-, 6-, and 12-h post phage administration, spleens were harvested, homogenized, and centrifuged. The titer of viable phage in the supernatant was then determined using the double‑layer plaque assay.

To evaluate the therapeutic efficacy of the mutant phage AbP20M in mice pre-exposed to AbP20, 6–8-week-old female BALB/c mice were immunized intramuscularly with 5 × 10¹⁰ PFU/mouse of AbP20. Two weeks after the last boost, mice were intraperitoneally challenged with 2 × 10⁸ CFU/mouse of A. baumannii Ab28 and then received intraperitoneal administration of AbP20M or the wild-type phage AbP20 at 2 × 10⁹ PFU/mouse (MOI = 10) or 2 × 10¹⁰ PFU/mouse (MOI = 100) at 1- and 8-h post‑infection. Survival was monitored every 12 h for 6 days (n = 6). At 24 h post‑infection, the liver, kidney, and spleen were harvested from each group for CFU enumeration (n = 4). Three days before infection, serum from each group was collected via retro‑orbital bleeding for determination of antibody titers and neutralizing activity.

Blood routine examination

Blood collected in microcentrifuge tubes containing EDTA-K2 was gently mixed 8–10 times by inversion to ensure homogeneity. Hematological analysis was subsequently performed using an automated hematology analyzer (HEMAVET 950FS).

Histopathological examination

Mouse tissues were collected, fixed in 4% paraformaldehyde, and embedded in paraffin. The paraffin-embedded tissues were then sectioned at 5 μm thickness and stained with hematoxylin and eosin for histological examination. The stained sections were then observed and photographed using a digital image analysis platform 3DHISTECH (Hungary).

Purification of IgG from serum

Serum was diluted 1:5 with Binding/Wash buffer (20 mM disodium hydrogen phosphate, 0.15 M sodium chloride, pH 7.0). A 1.5 mL aliquot of Protein G resin was added to a chromatography column preloaded with 1.5 mL of Binding/Wash buffer. Before sample loading, the column was equilibrated with 5 mL of Binding/Wash buffer. The diluted serum was then loaded and allowed to flow out slowly. After washing with 30 mL of Binding/Wash buffer, bound IgG was eluted using Elution buffer (0.1 M glycine, pH 2.5). One‑10th volume of Neutralization buffer (1 M Tris‑HCl, pH 8.5) was added to the eluate to adjust the pH to 7.4. The antibody concentration was determined, and the purified IgG was aliquoted and stored at −80 °C until use.

Preparation of IgG Fab fragments

The prepared IgG was dialyzed against 0.01 M PBS (pH 7.4) for 3 h, and the antibody concentration was adjusted to 1 mg/mL. Papain powder was dissolved in water to prepare a 10 mg/mL stock solution, which was filtered through a 0.22 μm filter and stored at 4 °C. The enzyme was diluted to 1 mg/mL in a solution containing 1.1 mM EDTA, 0.067 mM β‑mercaptoethanol, and 5.5 mM cysteine‑HCl, and then incubated for 30 min for activation. Papain and the antibody solution were mixed at a ratio of 1:10 and incubated at 37 °C for 12 h. The reaction was stopped by adding 33 mM N‑ethylmaleimide. The reaction product was ultrafiltrated using a 30 kDa ultrafiltration tube to remove papain and exchange the buffer to 0.01 M PBS (pH 7.4). The digested product was passed through a Protein G resin, and the flow‑through fraction was collected to remove Fc fragments. Each fraction was verified by SDS‑PAGE.

Serum neutralization assay

Ten μL of 2 × 10¹⁰ PFU/mL phage AbP20 was mixed with 2 μL of varied immune serum or purified IgG, and 88 μL of PBS and then incubated at 37 °C for 1 h. The phage‑serum mixture was then serially diluted in PBS and titrated using the double‑layer plaque assay. The phage neutralization efficacy of each serum was presented as efficiency of plating (EOP) that calculated as the ratio of the phage titer in each treatment to the titer in the serum‑free control.

ELISA assay

Phage AbP20 or individual phage structural proteins were diluted in 100 μL of coating buffer (bicarbonate‑carbonate, pH 9.6) and coated at 2 × 10⁹ PFU/well of phage or 500 ng/well of protein in 96-well plates (Corning) overnight at 4 °C. After washing four times with 300 μL of PBST (PBS containing 0.05% Tween‑20), plates were incubated with 5% skim milk in TBST for 2 h at 37 °C. Subsequently, 100 μL of diluted immune serum was added and incubated at 37 °C for 2 h. Following four washes with 300 μL PBST, plates were supplemented with 100 μL/well of HRP‑conjugated goat anti‑mouse IgG or its subtypes (1:10,000 diluted) and incubated at 37 °C for 1 h. After washing another four times with PBST, each well was incubated with 100 μL of 3,3′,5,5′‑tetramethylbenzidine substrate for 15 min at room temperature and then supplemented with 2 M H2₂SO₄. The endpoint titer of immune serum was defined as the highest dilution fold that yielded an absorbance at 450 nm more than 2.1‑fold of the mean value from naïve mouse serum-treated groups.

Determination of the neutralization stage

To determine the effect of each structural protein-elicited serum on phage adsorption, serum diluted 100‑fold were incubated with 10⁸ PFU of AbP20 at 37 °C for 1 h, followed by incubation with 10⁸ CFU/mL of Ab28 at 4 °C for 2 h. After five washes with PBS, samples were analyzed by Western blotting. Gray values were calculated using ImageJ software. To determine the effect of serum against different components of AbP20 on phage DNA injection, sera diluted 100‑fold were incubated with 10⁸ PFU of AbP20 at 37 °C for 1 h, followed by incubation with 10⁸ CFU/mL of Ab28 at 37 °C for 20 min. After five washes with PBS, the copies were determined by qPCR as described below, and the inhibition rate was calculated. To determine the effect of serum against different components of AbP20 on phage release, sera diluted 100‑fold were incubated with 10⁸ PFU of AbP20 at 37 °C for 1 h, followed by incubation with 10⁸ CFU/mL of Ab28 at 37 °C for 2 h. After centrifugation, the supernatant was collected, and phage titers were determined using the double‑layer agar plate method.

Phage nucleic acid extraction and qPCR detection

Phage nucleic acids were extracted from bacterial samples using the FineMag Rapid Magnetic Bead Method Virus DNA/RNA Extraction Kit (GENFINE) according to the manufacturer's instructions. Quantitative polymerase chain reaction (qPCR) was performed with the Hieff SYBR Green qPCR Master Mix (YEASEN). All qPCR assays were carried out on a Bio-Rad CFX96 instrument (Bio-Rad). The threshold cycle (Ct) value for each sample was determined using Bio-Rad CFX Manager 3.1 software. The RNA polymerase β subunit gene fragment of Ab28 was used as an internal control. The qPCR primer sequences are listed in S1 Table.

Polyclonal rabbit anti-gp37 antibodies

The gp37 protein was mixed with an aqueous rapid immunoadjuvant for rabbit immunization. Following the immunization, antiserum was collected and subjected to titer determination. Subsequently, 1 mg of the purified protein was covalently conjugated to a cyanogen bromide-activated Sepharose 4B column to prepare an affinity purification column and incubated with the antiserum overnight. After washing with HCl (pH 5.0) to remove nonspecific antibodies, column was eluted with 0.15 M Glycine-HCl buffer (pH 2.5), and the passthrough solution was immediately neutralized with 10 × PBS. The concentration of the purified antibody was measured using the Bradford assay, followed by further titer validation to ensure that their binding activity and specificity met experimental requirements.

Detection of phage phagocytosis in cell

RAW264.7 cells were seeded in 24-well plates at a density of 3 × 10⁵ cells per well for 12 h, after replacing the medium with DMEM containing 2% FBS, each well was supplemented with 10 μL of 1 × 10¹⁰ PFU AbP20, 85 μL of PBS, and 5 μL of varied immune sera, purified IgGs or their Fab fragments, and then incubated for 6 h at 37 °C with 5% CO₂. Following incubation, the cells were washed with PBS (pH 7.4) and lysed by RIPA lysis buffer (Beyotime) supplemented with protease inhibitors (Roche). Cell lysates were then mixed with 5 × loading buffer, boiled at 100 °C for 10 min, and then analyzed by SDS‑PAGE. Resolved protein bands were transferred onto a polyvinylidene fluoride membrane (Millipore) and blocked with 5% skimmed milk in PBST 1 h at room temperature, followed by overnight incubation with rabbit polyclonal anti-gp37 antibodies at 4 °C. After four washes with PBST, the membrane was incubated with horseradish peroxidase-conjugated anti‑rabbit IgG (Proteintech). Protein bands were visualized using an enhanced chemiluminescence kit (Millipore). The band intensity of gp37 from each group was quantified by Image J and compared to that of the PBS-immunized group (Vehicle). All experiments were carried out by triplicates taking GAPDH as reference.

Fc receptor blocking assay

RAW264.7 macrophages were seeded in 24-well plates at a density of 3 × 10⁵ cells per well and cultured for 12 h. Afterwards, the culture medium was replaced with DMEM supplemented with 2% FBS, followed by the addition of MonoZero Mouse Fc Blocking Reagent (Proteintech) at a dosage of 0.5 μg per 100 μL cell suspension. Following gentle pipette mixing, cells were incubated at 37 °C for 20 min. The premixed phage-serum sample was subsequently added to each well, and co-cultured with cells at 37 °C for 6 h. Finally, cellular samples were harvested for subsequent western blot analysis.

Immunofluorescence assay

RAW264.7 cells were seeded in 24-well plates at 2 × 10⁵ cells per well for 12 h, after replacing the medium with DMEM containing 2% FBS, each well was supplemented with 10 μL of 1 × 10¹⁰ PFU AbP20, 85 μL of PBS, and 5 μL of varied immune serum and then incubated for 6 h at 37 °C with 5% CO₂. Subsequently, wells were washed five times with PBS to remove uninternalized phage and then fixed with 4% paraformaldehyde for 15 min. Following three times washing with PBS, cells were permeabilized with 0.1% Triton X-100 for 10 min and followed by another three PBS washes before blocking with blocking solution (Beyotime) for 30 min. Subsequently, cells were incubated overnight at 4 °C with a rabbit polyclonal anti-gp37 antibodies (1:200 diluted). After three washes with PBS, cells were incubated for 1 h at room temperature in dark with a CoraLite488-conjugated Goat Anti-Mouse IgG(H + L) secondary antibody (1:200 diluted). Following three additional washes with PBS, the cytoskeleton was stained using ActinRed 555 ReadyProbes Reagent (Thermo Fisher) for 30 min at room temperature. Finally, after three further PBS washes, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; Leagene) at a concentration of 1 μg/ml for 10 min at room temperature. Cells were then mounted with Antifade Mounting Medium (Beyotime) and imaged using a Leica STELLARIS 8 WLL confocal fluorescence microscope. The mean fluorescence intensity in each treatment was calculated by LAS X software from six distinct regions and compared to that of the group treated with PBS-immunized serum (Vehicle).

Detection of serum-mediated phage aggregation

To detect the size distribution of phage aggregates, 20 μL of 1 × 10¹⁰ PFU AbP20 was mixed with 170 μL of PBS and 10 μL of varied immune sera, purified IgGs, or their Fab fragments and incubated at 37 °C for 1 h. Subsequently, the diameter distribution of phage aggregates was measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Panalytical, UK). To capture the image of phage aggregates, 10 μL of 1 × 10¹⁰ PFU AbP20 was incubated with 88 μL of PBS and 2 μL of varied immune serum or IgGs at 37 °C for 1 h. Samples were then imaged by transmission electron microscopy following the previously described protocol.

In vitro phage evolution

AbP20-immune serum was diluted to a starting titer of 1:800, pre-incubated with phage AbP20 for 30 min, and subsequently co-cultured with A. baumannii strain Ab28. Following 6 h of co-incubation, culture supernatants were harvested via centrifugation. After gradient dilution and plaque purification, 10 independent plaques were randomly picked for serial passaging per selection round. To impose progressive immune selection pressure, the serum dilution ratio was reduced by 100-fold per round (from 1:800 sequentially to 1:700, 1:600 and ultimately 1:100), and the 1:100 serum dilution group was subjected to triple independent screening. Starting from the 4th selection round, efficiency of plating (EOP) assays were performed to assess the neutralization escape capacity of evolved phage variants. Post-selection phages were purified and amplified from single plaques in each round. The full-length coding sequences of gp40 and gp44 were amplified by PCR and subjected to sequencing to screen for mutation sites.

Statistical analysis

Data were presented as mean ± SD. Statistical analysis was performed using GraphPad Prism (GraphPad Software). Statistical significance was calculated using a two-tailed Student t test. Survival curves were analyzed with Log-rank (Mantel-Cox) test by GraphPad Prism (GraphPad Software). ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

S1 Fig. Activity and genome characterization of phage AbP20.

(A) Bactericidal activity of AbP20 against A. baumannii Ab28. Exponential cultures of Ab28 were cultured in LB at 37 °C with shaking at 200 rpm in the presence of AbP20 at varied MOIs. Viable bacteria were quantified by enumerating the CFU at 0, 1, 2, 4, and 6 h of coculture. Dashed line indicates the limit of detection. (B) Genomic annotation of AbP20. The AbP20 genome was sequenced, and all predicted open reading frames are labeled. The data underlying this figure can be found in S1 Data.

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S2 Fig. Imbalance in peripheral blood cells is mitigated by high-dose AbP20 treatment.

Peripheral blood counts of total neutrophils, monocytes, and platelets were assessed in AbP20-treated group. According to the manufacturer's specifications, all measured values were determined to be within normal reference ranges. The data underlying this figure can be found in S1 Data.

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S3 Fig. Safety evaluation of phage AbP20.

(A) Body weight changes in mice after intraperitoneal administration of 1 × 10¹⁰ PFU/mouse AbP20 daily for 7 consecutive days. Body weight was measured daily for 10 days. (B) Total white blood cell, neutrophil, lymphocyte, monocyte counts in peripheral blood from AbP20-treated mice. All values were within the normal reference range as defined by the assay manufacturer's instructions. Dashed lines indicate the healthy range. (C) Histopathological evaluation. Organs were collected from mice 1 day after the final AbP20 treatment and assessed by hematoxylin and eosin staining. The data underlying this figure can be found in S1 Data.

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S4 Fig. Purification and immunogenicity evaluation of AbP20 structural proteins.

(A) Heterologous expression of AbP20 structural proteins in E. coli. (B) Immunogenicity of AbP20 and its structural proteins. IgG titers from each immune serum (Serum) against AbP20 or each of its structural protein (Antigen) were determined by ELISA. (C-F) The titers of IgG subclasses in each immune serum against AbP20 or each structural protein (antigen) were determined by ELISA. Dashed line indicates the limit of detection. The data underlying this figure can be found in S1 Data. The uncropped gels can be found in S1 Raw Images.

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S5 Fig. Effects of the serum from varied groups on the infective activity of AbP20.

(A) Schematic diagram of the neutralization assay. (B) Infective activity of AbP20 in the presence of varied immune serum. Phage AbP20 was incubated with varied immune serum diluted 50–800 folds at 37 °C for 1 h. The double-layer plaque assay was then used to evaluate the infective activity of AbP20. (C) Effect of serum against different components of AbP20 on phage adsorption. Serum diluted 100‑fold was incubated with 10⁸ PFU of AbP20 at 37 °C for 1 h, followed by incubation with 10⁸ CFU/mL of Ab28 at 4 °C for 2 h. After washing with PBS, the samples were analyzed by Western blotting. The uncropped blots and gels can be found in S1 Raw Images.

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S6 Fig. AbP20 immune serum increases macrophage-mediated phage phagocytosis.

(A) IFA of AbP20 phagocytosis by macrophages after 6 h incubation with AbP20 immune serum. Phage AbP20 is labeled by rabbit anti-gp37 antibody and CoraLite 488 dye (green), β-actin stained with ActinRed 555 (red), and the nucleus stained with DIPA (blue). Scale bar: 20 μm. (B) Quantitative analysis of the mean fluorescence intensity performed using LAS X software, with measurements taken from six distinct regions for statistical analysis. The data underlying this figure can be found in S1 Data.

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S7 Fig. Purified IgGs targeting AbP20, gp40, gp44, and gp37 retain strong neutralizing activity and promote phage phagocytosis.

(A) Immunogenicity of purified antibodies. Sera were purified using Protein G resin to obtain purified IgG antibodies. The titers of the purified IgGs targeting each structural protein against AbP20 were determined by ELISA. (B) Effects of varied IgG on the efficiency of plating (EOP) of phage AbP20. (C) The effect of purified IgGs on AbP20 phagocytosis was assessed by western blot. Intracellular phages were detected using rabbit anti‑gp37 antibody, with Tubulin serving as a reference. Band intensities of gp37 from three independent Western blots were analyzed by Image J software and compared with those of the PBS‑treated control group. Dashed line indicates the limit of detection. Unpaired Student’s t tests. ns: not significant, *p < 0.05 and **p < 0.01. The data underlying this figure can be found in S1 Data. The uncropped blots can be found in S1 Raw Images.

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S8 Fig. Different IgG antibodies induce aggregation of phages.

Representative images of AbP20 aggregates in the presence of varied IgGs. Model manners of phage conjugation under each condition are presented above each image. Scalar bar: 100 nm.

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S9 Fig. AbP20M mirrors AbP20 in lytic curve, phagocytosis, and in vivo clearance.

(A) Inhibitory effects of AbP20M on the growth of A. baumannii 28. Exponential cultures of Ab28 were diluted with LB to an OD600 of 0.4 and incubated with different MOIs (0, 0.01, 0.1, 1, and 10) of AbP20 at 37 °C with shaking at 180 rpm. The turbidity of bacterial culture at 600 nm was monitored every 30 min for 12 h. (B) Phagocytosis of AbP20M by macrophages was detected by western blot. Intracellular phages were detected using a rabbit anti‑gp37 antibody, with tubulin serving as a reference. Band intensities of gp37 from three independent Western blots were analyzed using ImageJ software. (C) Evaluation of the effects of antibodies on the clearance of phage AbP20M in vivo. Group immunized with PBS served as control (Vehicle). All mice received a single dose of AbP20 intraperitoneally 14 days post the last boost. Spleens were collected at 1-, 3-, 6-, and 12-h post-injection for phage titer determination (n = 3). Unpaired Student’s t tests. ns: not significant. The data underlying this figure can be found in S1 Data. The uncropped blots can be found in S1 Raw Images.

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S1 Raw Images. All the uncropped western blot and SDS-PAGE images contained in the manuscript.

Uncropped western blot images corresponding to Figs 3B, 3D, 5A, 5B, 5E, 5F, 5G, 5H, 6F–6H, S4A, S5C, S7C, and S9B.

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S1 Data. The numerical data underlying Figs 1B, 1D–1G, 1I–1L, 2C–2J, 3C, 3E, 3G, 3I, 4A, 5A, 5C–5H, 6C, 6E–6H, 6J–6M, S1A, S2, S3A, S3B, S4B–S4F, S6B, S7C, and S9A–S9C.

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