Novel, non-colonizing, single-strain live biotherapeutic product ADS024 protects against Clostridioides difficile infection challenge in vivo

Figure 1 Quantification of ADS024 (fresh culture) in the mice and miniature swine fecal samples for the single-dose study. A: The mice were predosed with vancomycin to mimic disruption of gut microbiota and administered a single oral dose of 5 × 10⁸ colony-forming units (CFU)/mouse of ADS024. Microbiological analysis was subsequently conducted on the mouse fecal pellets and gastrointestinal tissue. The ADS024 colonies were recovered in feces following plating on agar media at 4 h, 8 h, and 24-h post dose; B: The single-dose study in miniature swine included 3 groups predosed with vancomycin to induce disruption of gut microbiota. They were exposed to the following dose range of ADS024: Group 2 (low dose) 6.2 × 10⁷ CFU; Group 3 (medium dose) 7.1 × 10⁸ CFU; and Group 4 (high dose) 7.1 × 10⁹ CFU. There was 1 group not pre-dosed with vancomycin but exposed to a single dose of ADS024: Group 5 (no vancomycin, high dose) 7.1 × 10⁹ CFU. The control group (Group 1, placebo) did not receive vancomycin or ADS024. After single oral administration of ADS024 to the miniature swine, the ADS024 colonies, assessed by quantitative polymerase chain reaction (qPCR), were detected on agar plates in the ADS024-treated Groups 4 and 5, regardless of vancomycin therapy, at colony counts higher than placebo controls that displayed only Bacillus-like colonies and not ADS024-specific ones based on qPCR, at the 7-h to 48-h time points. P value significance: ^aP ≤ 0.05, ^bP ≤ 0.01, ^eP ≤ 0.001.

Figure 2 Clostridioidesdifficile infection challenge mouse model studies. Study 1: The effects of fresh (vegetative) ADS024 culture vs ADS024 spore preparations on the clinical manifestations of Clostridioides difficile infection (CDI) over 10 d. ADS024 was delivered to the mice (n = 15 per group) as resuspended spore suspensions [in phosphate-buffered saline (PBS)] or as a freshly prepared daily culture (ADS024 bacteria resuspended in PBS) 24 h prior to C. difficile. A: The weight loss comparison between mice receiving vegetative vs spore ADS024 relative to the infection (vehicle-dosed) controls; B: The adverse health effects comparison in surviving animals among the 3 groups (control, vegetative ADS024, and spore ADS024); C: The daily score of adverse health observations among the 3 groups was determined as shown in Supplementary Table 1. Study 2: The effects of single daily (QD) doses vs 3 times daily (TID) administration with fresh ADS024 in the mice (n = 10 per group) over the course of 10 d; D: The weight loss comparison among the 4 groups (vancomycin, untreated, QD, and TID); E: The adverse health effects comparison in the surviving animals among the 4 groups; F: The daily score of adverse health observations among the 4 groups was determined as shown in Supplementary Table 1.

Figure 3 Distal colon model. A: Comparison of the wells inoculated with ADS024 vs media alone was done via quantitative polymerase chain reaction (qPCR) using specific primers to detect ADS024 as described in Materials and Methods; B: qPCR was performed to study the antimicrobial activity of ADS024 on 5 different phyla of the human colonic microbiota; C: MiSeq compositional sequencing analysis was performed to compare the impact of ADS024 on the gut microbiota at the phylum level with that of media alone; D: MiSeq was used to determine the ADS024 effect after 24 h of treatment at the genus level. ^eP ≤ 0.001.

Figure 4 Distal colon model: Alpha and beta diversity analyses. MiSeq compositional sequencing and bioinformatic analysis were performed to measure the microbiota diversity changes following ADS024 treatment using the: A: Shannon index for alpha diversity; B: Simpson diversity index for alpha diversity; C: UniFrac principal coordinates analysis for beta diversity.

Figure 5 The impact of ADS024 on microbiota diversity in the miniature swine fecal samples during the 28-d study. MiSeq compositional sequencing and bioinformatic analysis were performed to measure the impact of ADS024 on the microbiota in miniature swine fecal samples after 28 d using the: A: Shannon index for alpha diversity; B: Simpson diversity index for alpha diversity; C: UniFrac principal coordinates analysis (PCoA) for beta diversity.

Figure 6 Effect of ADS024 on the gut microbiota of miniature swine during the 28-d study at the phylum and genus levels. A and B: MiSeq compositional sequencing and bioinformatic analysis was performed to measure the impact of ADS024 on the gut microbiota of miniature swine at the (A) phylum and (B) genus levels.