Research ArticleInfectious disease Open Access | 10.1172/jci.insight.181309

Arne Echterhof,1,2 Tejas Dharmaraj,1 Arya Khosravi,1 Robert McBride,3 Lynn Miesel,4 Ju-Hsin Chia,4 Patrick M. Blankenberg,1 Kun-Yuan Lin,4 Chien-Chang Shen,4 Yu-Ling Lee,4 Yu-Chuan Yeh,4 Wei Ting Liao,4 Francis G. Blankenberg,5 Krystyna Dąbrowska,6 Derek F. Amanatullah,7 Adam R. Frymoyer,8 and Paul L. Bollyky1

1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

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1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

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1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

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1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

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1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

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1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

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1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

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1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

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1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

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1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

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1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

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1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

Find articles by Liao, W. in: JCI | PubMed | Google Scholar

1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

Find articles by Blankenberg, F. in: JCI | PubMed | Google Scholar

1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

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1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

Find articles by Amanatullah, D. in: JCI | PubMed | Google Scholar

1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

Find articles by Frymoyer, A. in: JCI | PubMed | Google Scholar

1Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.

2Institute of Medical Microbiology, University Hospital of Muenster, Muenster, Germany.

3Felix Biotechnology, South San Francisco, California, USA.

4Pharmacology Discovery Services, Taipei, Taiwan.

5Division of Pediatric Radiology and Nuclear Medicine, Department of Radiology, Lucile Packard Children’s Hospital, Stanford, California, USA.

6Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

7Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California, USA.

8Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA.

Address correspondence to: Paul L. Bollyky, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center for Molecular and Genetic Medicine, 279 Campus Drive, Stanford, California 94305, USA. Email: pbollyky@stanford.edu.

Authorship note: AE, TD, and AK are co–first authors.

Find articles by Bollyky, P. in: JCI | PubMed | Google Scholar

Authorship note: AE, TD, and AK are co–first authors.

Published October 22, 2024 - More info

Published in Volume 9, Issue 20 on October 22, 2024
JCI Insight. 2024;9(20):e181309. https://doi.org/10.1172/jci.insight.181309.
© 2024 Echterhof et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published October 22, 2024 - Version history
Received: April 4, 2024; Accepted: September 10, 2024 View PDF Abstract

With the increasing prevalence of antimicrobial-resistant bacterial infections, there is interest in using bacteriophages (phages) to treat such infections. However, the factors that govern bacteriophage pharmacokinetics in vivo remain poorly understood. Here, we have examined the contribution of neutrophils, the most abundant phagocytes in the body, to the pharmacokinetics of i.v. administered bacteriophage in uninfected mice. A single dose of LPS-5, a bacteriophage recently used in human clinical trials to treat drug-resistant Pseudomonas aeruginosa, was administered i.v. to both immunocompetent BALB/c and neutropenic CD1 mice. Phage concentrations were assessed in peripheral blood and spleen at 0.25, 1, 2, 4, 8, 12, and 24 hours after administration by plaque assay and qPCR. We observed that the phage clearance was only minimally affected by neutropenia. Indeed, the half-lives of phages in blood in BALB/c and CD1 mice were 3.45 and 3.66 hours, respectively. These data suggest that neutrophil-mediated phagocytosis is not a major determinant of phage clearance. Conversely, we observed a substantial discrepancy in circulating phage levels over time when measured by qPCR versus plaque assay, suggesting that significant inactivation of circulating phages occurs over time. These data indicate that alternative factors, but not neutrophils, inactivate i.v. administered phages.

Introduction

The global crisis of bacterial antimicrobial resistance (AMR) represents a profound challenge to human health. Once-treatable infections are now responsible for extensive morbidity and mortality and have the potential to evolve into global health problems of pandemic dimensions (1).

Pseudomonas aeruginosa is one of the most problematic pathogens for which new treatment options are needed (2–4). Multidrug-resistant (MDR) strains of P. aeruginosa are prevalent in environmental and clinical settings due to acquired resistance and intrinsic mechanisms of resistance (5, 6). There is a need for innovative treatments to treat MDR P. aeruginosa and other pathogens.

Bacteriophages (phages), viruses that kill bacteria, are an exciting treatment option for AMR bacterial infections (7–10). Multiple studies have demonstrated the efficacy of phage therapy against MDR P. aeruginosa (11–14), either alone or in conjunction with conventional antibiotics (15). Phage therapy is saving lives; however, success has been inconsistent (15–18). This has limited the therapeutic and commercial prospects of this approach.

Critical to the successful development of phages as a therapeutic treatment will be a deep understanding of its pharmacokinetics (PK) (19). While there is, in fact, a large body of literature on phage therapy, it is heterogenous; difficult to access, as much of it is in the non-English-language literature; and often predates the current era of phage therapy. Fortunately, this literature has been summarized in a recent review (20).

A common method of delivering therapeutic phages is intravenous (i.v.) administration, particularly for systemic and pulmonary infections (21). After i.v. administration, the half-life (t1/2) of phages in the blood of mice has been reported as between 2.2 hours and 4.5 hours in different animal models (22, 23). Most phages are cleared from the blood by the liver and spleen within minutes to hours (24–26), and i.v. administration of phages leads to phage accumulation in these tissues. The rapid removal of phages from circulation and subsequent degradation limits their potential therapeutic efficacy (27).

Endocytosis plays a prominent role in the clearance of many viruses and foreign molecules. Large viral particles exit the circulation and enter the liver and splenic sinusoids, where a high density of professional phagocytes is present (24, 27) in the mononuclear phagocyte system, also known as the reticuloendothelial system. Phages may also exert pharmacodynamic effects by influencing the inflammatory properties of mononuclear cells (23, 28, 29). However, neutrophils are also abundant in the liver, spleen, and peripheral tissues and are the most common phagocyte in circulation. Neutrophils have been reported to engulf viruses (30, 31). However, the contributions of neutrophils to phage clearance remains unclear.

In this study, we evaluated the PK of i.v. administered P. aeruginosa phage LPS-5 in an immunocompetent and neutropenic mouse model.

Results

Calibration studies to evaluate our ability to assess phage concentrations. We chose to study phage LPS-5, an antipseudomonal phage recently included in a human clinical trial of phage therapy, the CYstic Fibrosis bacterioPHage Study at Yale (CYPHY; ClinicalTrials.gov NCT04684641). LPS-5 is a member of the Pakpunavirus family and uses Pseudomonal LPS for viral entry (14). A representative transmission electron micrograph of LPS-5 and an image demonstrating LPS-5 plaque morphology on a lawn of P. aeruginosa PAO1 are shown in Figure 1.

Figure 1

LPS-5 is a tailed phage used in the treatment of drug-resistant infections. (A) A representative transmission electron microscopy image of LPS-5 (original magnification, ×80,000) is shown in extended and contracted states. (B) Lytic plaque morphology for LPS-5 on Pseudomonas aeruginosa strain PAO1.

We first sought to assess the linearity, accuracy, and precision of plaque assays and quantitative PCR (qPCR) to measure phages in sera and tissues collected from immunocompetent BALB/c and neutropenic CD1 mice over a broad range of phage concentrations. A known amount of LPS-5 (103, 104, 105, 106, 107, 108 PFU/sample) was added to PBS (saline), whole peripheral blood, or excised spleen tissue. Spleen tissues were homogenized after adding phages. These were then analyzed by plaque assays (Figure 2, A–C and G–I) or qPCR (Figure 2, D–F and J–L).

Figure 2

Calibration curves for phage titrations in blood and spleen. To assess our ability to accurately quantify phages in blood and tissue samples, we performed titration studies using plaque assays and qPCR assays as our readouts. For these studies phages with established, nominal concentrations were added to PBS, blood, or spleen tissue collected from (A–F) immunocompetent BALB/c mice or (G–L) neutropenic ICR mice. These were then analyzed using (A–C and G–I) plaque assays or (D–F and J–L) qPCR. Log-transformed phage titers and Ct values are plotted over log-transformed normal phage titers. A linear regression was performed.

For the qPCR studies, the median accuracy in BALB/c samples was determined to be 101% (range, 90%–115%) for spleen and 93% (range, 88%–94%) for blood, and the uncertainty was determined to be 6.4% for spleen and 0.7% for blood. The median accuracy in CD1 samples was determined to be 88% (range, 85%–91%) for spleen and 100% (range, 100%–102%) for blood, and the uncertainty was determined to be 2.7% for spleen and 0.8% for blood. The limit of detection was determined to be 200 PFU/sample. The lower limit of qualification (LLOQ) was determined to be 1,000 PFU/sample. These data are strong indications of excellent linearity and reproducibility using these approaches.

The PK of phage LPS-5 in immunocompetent BALB/c mice. Having established methods to assess phage levels in mouse tissues, we next sought to determine the PK of phage LPS-5 in immunocompetent mice. For these studies, we used BALB/c mice, a well-established mouse strain commonly used in pharmacology studies.

Using these animals, we examined phage PK using the approach shown in Figure 3. LPS-5 phage was administered by i.v. tail vein injection at a single dose (0.1 mL/mouse at a concentration of 1.0 × 1011 PFU/mL) to BALB/c mice. Animals were sacrificed after 0.25 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours. Phage concentrations were measured in whole blood and spleen homogenates (Figure 4, B and D) using plaque assays and qPCR. The concentration over time was graphed and noncompartmental analysis was performed.

Figure 3

Schematic of biodistribution and pharmacokinetics experiment. In these studies, phage LPS-5 was administered i.v. as a single dose (0.1 mL/mouse at 1.0 × 1011 PFU/mL) to immunocompetent BALB/c mice or neutropenic CD1 mice. Animals were sacrificed after 0.25 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours. At every sampling time point, 5 mice were sacrificed, and the phage concentration was measured in whole blood and spleen homogenates by qPCR and plaque assay. Noncompartmental analysis was conducted to estimate pharmacokinetic parameters.

Figure 4

Pharmacokinetics and biodistribution of LPS-5 bacteriophage in blood and spleen tissue over time in BALB/c (immunocompetent) and CD1 (neutropenic) mice. Mice received 0.1 mL of 1.0 × 1011 PFU/mL LPS-5 phage suspension i.v. by tail vein injection. Five mice were sacrificed at 0.25, 1, 2, 4, 8, 12, and 24 hours for measurement by qPCR and plaque assays of blood and spleen tissue homogenates. 35 immunocompetent mice and 35 neutropenic mice were used. Geometric mean ± SD is plotted over time. (A–D) Blood pharmacokinetics (A and B) and spleen biodistribution (C and D) of LPS-5 in immunocompetent BALB/c versus neutropenic CD1 mice, as determined by qPCR (A and C) or spot assay (B and D). (E–H) Comparison of PK curves derived from qPCR and spot assay in blood (E and F) and spleen (G and H) in immunocompetent BALB/c (E and G) and neutropenic CD1 mice (F and H). The dotted lines indicate lower limit of detection (LLOD). **P < 0.001, ***P < 0.0001, ****P < 0.00001, t tests adjusted for multiple comparisons using Holm-Šídák method. Statistical analyses were performed on means of log-transformed values due to right-skewness of PK data.

The concentration-time profile, as measured by plaque assay of active LPS-5 after i.v. administration in conventional BALB/c mice, is shown in Figure 4A. The Cmax of active phages in blood in BALB/c mice was 8.55 × 105 PFU/mL, approximately 0.01% of the injected dose, suggesting extremely rapid clearance (Figure 4B) of phages from the bloodstream. Circulating active phages demonstrated first-order elimination with a terminal t1/2 of 3.24 hours. By 24 hours, very low concentrations of active phage remained in the blood (C24 = 539 PFU/mL). High concentrations of active phages were rapidly achieved in spleen tissue (Cmax = 2.44 × 108 PFU/g) and remained relatively high over the 24-hour period of measurement (C24 = 4.91 × 106 PFU/g) (Figure 4C). Other pharmacokinetic parameters are listed in Table 1.

Table 1

Pharmacokinetic parameters for immunocompetent BALB/c and neutropenic CD1 mice in blood and spleen based on qPCR and plaque assay

Together, these data suggest that bacteriophages leave the circulation rapidly after administration and accumulate in filtration organs, including the spleen. These data suggest that the phage is removed from the bloodstream by the mononuclear phagocyte system.

The PK of phage LPS-5 in neutrophil-deficient mice. We then asked how neutropenia affected phage PK and uptake by the spleen. For these studies, we used the well-established CD1 neutropenic mouse model (32, 33).

Here again, LPS-5 phage was administered by i.v. tail vein injection at a single dose (0.1 mL/mouse with a concentration of 1.0 × 1011 PFU/mL) to CD1 mice. Animals were then sacrificed at various time points, and phage concentrations were measured in whole blood and spleen homogenates.

In general, we observed a highly similar PK profile in CD1 mice compared with BALB/c mice (Figure 4, E–H). The Cmax of phages in blood in CD1 mice was 3.75 × 106 PFU/mL, approximately 0.05% of the injected dose, again suggesting extremely rapid clearance. C24 was low, at 249 PFU/mL. As with the BALB/c mice, we once again observed first-order elimination of active phage particles in blood from CD1 mice, with a t1/2 in the blood of 3.94 hours (Figure 4C). The concentration of active viral particles decreased in spleen tissue by approximately 1 log over the measured duration, with a Cmax in the spleens of 4.05 × 108 PFU/g and a C24 of 4.33 × 107 PFU/g (Figure 4D). Other pharmacokinetic parameters are listed in Table 1.

As with the BALB/c mice, these data indicate that in neutropenic CD1 mice, LPS-5 bacteriophages leave the circulation rapidly after administration and accumulate in other compartments like the spleen.

In comparing the values for neutropenic CD1 mice to those for conventional BALB/c mice, we note that the values for Cmax, C24, and t1/2 are similar among these strains (Figure 4, E–H). For example, the t1/2 of phages in blood in BALB/c and CD1 mice is 3.24 hours and 3.94 hours, respectively. There was a relatively modest 3- to 4-fold increase in the AUC from the time of dosing extrapolated to infinity (AUCinf) in the blood and spleen tissue of neutropenic mice, but the overall PK are similar between the two groups of animals. These data suggest that neutrophil-mediated phagocytosis is not the major determinant of phage clearance.

Phages are inactivated in circulation. Our data reveal a substantial discrepancy in phage quantity over time when measured by qPCR versus plaque assay in blood, especially at later time points. The C24 in the blood of the BALB/c mice measured by qPCR was 1.89 × 104 PFU/mL versus 539 PFU/mL measured by plaque assay. This is a difference of around 35-fold. In the CD1 mice, the C24 was 2,160 PFU/mL measured by qPCR versus 249 measured by plaque assay. This is equivalent to a 9-fold difference, approximately (Table 1).

These data indicate that factors in circulation reduce the number of functional phages (measured by plaque assay) compared with the number of functional phages plus phage fragments (measured by qPCR). Phages stay active for longer in the spleen than in the blood. The AUC24 in the spleen of the BALB/c mice measured by plaque assay was