Phage therapy with nebulized cocktail BX004-A for chronic Pseudomonas aeruginosa infections in cystic fibrosis: a randomized first-in-human trial

Abstract

Cystic fibrosis is a monogenetic disease complicated by recurrent bacterial lung infections that require chronic antibiotics. Pseudomonas aeruginosa is an increasingly antibiotic-resistant pathogen associated with cystic fibrosis morbidity and mortality. Here, we describe the development of a three-phage cocktail (BX004-A) designed to target a wide range of P. aeruginosa strains. We evaluated BX004-A in Part 1 of a first-in-human double-blind placebo-controlled phase 1b/2a clinical trial, which included nine adult cystic fibrosis patients chronically infected with P. aeruginosa (NCT05010577). BX004-A met the primary endpoints of safety and tolerability. Exploratory endpoints included pharmacokinetics and Pseudomonas aeruginosa sputum density reduction. Efficient phage delivery to the lower respiratory tract was observed, and a potential reduction in P. aeruginosa sputum burden was noted in the phage arm. However, due to the study's small sample size, definitive conclusions regarding efficacy are limited. These data pave the way toward further development of novel phage-based therapeutics in antibiotic-resistant pulmonary bacterial infections.

Fig. 1. Genomic analysis of the P. aeruginosa clinical isolate panel.

A Tree visualization of genomic distances between RefSeq P. aeruginosa cluster representatives and 143 clinical isolates in our panel, marked with red lines. B Scatter plot depicting the correlation between aPDS prevalence in our panel and that of 335 complete RefSeq genomes. Spearman correlation was applied, p-value (two-sided)= 4.093e-24 (C) Prevalence of aPDS in our panel by descending order. The color scheme represents the prevalence of aPDS across 335 P. aeruginosa complete RefSeq genomes. Source data are provided as a Source Data file.

Fig. 2. P. aeruginosa transcriptomic profiling.

A Illustration of distance metric applied in (B, C, and D). B, C, D Each panel depicts Euclidean distances of PCA coordinates within (four left boxes) and between (three right boxes) groups. Each dot represents a distance between two coordinates. The tested groups were BHIS solid (red), BHIS liquid (yellow), SCFM (gray) and fresh sputum (blue). Two sets of p-values are given for each analysis: (I) two sided Mann–Whitney U test (MW) testing the null hypothesis that distance distributions within groups are similar to distances between groups, and a Kruskal-Wallis one-way analysis of variance (KW) testing the null hypothesis that distances between all groups are similarly distributed. B PCA conducted for transcriptomic profiles of all P. aeruginosa core genes. C PCA conducted for transcriptomic profiles of all P. aeruginosa genes known to play a role in synthesis of phage binding molecules on the bacterial cell surface. D PCA for transcriptomic profiles of all P. aeruginosa genes involved in the alginate biosynthesis pathway. B–D Each datapoint in the figure represents a distance between two biological repeats. Sample sizes were: human sputum–20, SCFM–27, Solid–8, Liquid–8 (see Supplementary Data 4 for further details). Boxplots represent median (bold horizontal central line), quartiles 0.25 and 0.75 (box edges), minimum and maximum (whiskers). Source data are provided as a Source Data file.

Fig. 3. BX004-A anti-P. aeruginosa phage cocktail.

A Transmission electron microscopy (TEM) image of phage BMX-P1 revealed an icosahedral head (~55 nm x 52 nm) and short tail (estimated 14 nm × 11 nm). B Annotation of phage BMX-P1’s 44,725 base-pairs long circular genome. BMX-P1 belongs to the genus Bruynoghevirus. C TEM image of phage BMX-P2 revealed an icosahedral head (71 nm × 66 nm) and long contractile tail (rough estimation 131 nm × 18 nm). D Annotation of phage BMX-P2’s 65,780 base-pairs long circular genome. BMX-P2 belongs to the genus Pbunavirus. E TEM image of phage BMX-P3 revealed an icosahedral head (70 nm × 64 nm) and rigid contractile 119 nm long tail. F Annotation of phage BMX-P3’s 90,869 base-pairs long circular genome. BMX-P3 belongs to the genus Pakpunavirus. Micrographs are representative images from 10 or more repeats, see “Methods” section for further information.

Fig. 4. Characterization and performance of BX004-A.

A Schematic illustration of P. aeruginosa genes involved in phage life cycle, as discovered by our analysis. Phage are color-coded as depicted on the top right legend. The arrows by the gene names represent the average transcript abundance rank of the gene in sputum of pwCF (→: percentile 0.33–0.66, ↑: percentile > 0.66). This graphic was created in BioRender. Weiner, I. (https://BioRender.com/9qfpmc8). B Tree visualization of genomic distances between RefSeq P. aeruginosa cluster representatives and the 143 clinical isolates in our panel. Strain sensitivity to BX004-A is given in the outer circles. C Subpopulation analysis of BX004-A host range on strains containing each of the prevalent P. aeruginosa aPDS (found in at least 5% of strains). Enrichment tests – using Fisher’s exact test with Benjamini Hochberg correction, returned p > 0.05 in all cases. D, E Antibiofilm efficacy, as measured by ATP quantitation (D) and biomass staining (E). The two different strains used in this experiment are color-coded as depicted on the top right corner of panel D. Data shown for each condition is average ± SE across all available biological repeats. Sample sizes per condition alongside all other source data are provided as a Source Data file.

Fig. 5. Development of nebulized phage formulation.

A Experimental setup of aerosol collection system comprised of a general use mesh nebulizer (right), respiratory low resistance filter (middle), and breathing simulator BRS1100 (left). B Phage relative abundance as measured by quantitative PCR using phage-specific primers and probes, across all stages of a Next Generation Impactor. Source data are provided as a Source Data file.

Fig. 6. Phase 1b/2a clinical trial.

A–D Subjects receiving BX004-A are marked in blue, whereas subjects receiving placebo are marked in orange. Semi-transparent curves represent individual patients, whereas bold lines represent mean ± SE of the group. On day 3, there were two collection timepoints, marked 3a and 3b. Seven patients received BX004-A whereas two patients received placebo. Treatment duration (days 1–8) is indicated by gray shading. A Schematic illustration of the trial design is given above the panel (QD: daily; BID: twice daily), followed by a pharmacokinetics curve. Y-axis values of zero represent no detectable phages in sample. B Pharmacodynamic curves depicting change in P. aeruginosa sputum density over the course of treatment. Y-axis values of zero represent no change in CFU compared to baseline. The p-values (two-sided t-test) are day2:9e-4, day3a:0.045, day3b:0.780, day4:0.035, day8:0.486, day15:0.029. An earlier version of this pharmacodynamic analysis is presented in Table S8. C Relative abundance of P. aeruginosa within the sputum microbiome over time, as measured by 16S rRNA sequencing. The P-values (two-sided t-test) are day1:0.657, day8:0.411, day15:0.207. D Shannon diversity index of sputum microbiome over time, as measured by 16S rRNA sequencing. The p-values (two-sided t-test) are day1:0.940, day8:0.361, day15:0.121. Source data are provided as a Source Data file.