Development of safe and effective bacteriophage-mediated therapies against C. difficile infections a proof-of-concept preclinical study

Rasmussen, T. S.; Forster, S.; Larsen, S. B.; von Munchow, A.; Tranaes, K. D.; Brunse, A.; Castro-Mejia, J. L.; Adamberg, S.; Hansen, A. K.; Adamberg, K.; Hansen, C. H. F.; Nielsen, D. S.

doi:10.1101/2023.03.17.532897

bioRxiv · 10.1101/2023.03.17.532897

Development of safe and effective bacteriophage-mediated therapies against C. difficile infections a proof-of-concept preclinical study

Rasmussen, T. S.·Forster, S.·Larsen, S. B.·von Munchow, A.·Tranaes, K. D.·Brunse, A.·Castro-Mejia, J. L.·Adamberg, S.·Hansen, A. K.·Adamberg, K.·Hansen, C. H. F.·Nielsen, D. S.

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Abstract

BackgroundFecal microbiota transplantation (FMT) and fecal virome transplantation (FVT, sterile filtrated donor feces) have been effective in treating recurrent Clostridioides difficile infections, possibly through bacteriophage-mediated modulation of the gut microbiome. However, challenges like donor variability, costly screening, coupled with concerns over pathogen transfer (incl. eukaryotic viruses) with FMT or FVT hinders their wider clinical application in treating less acute diseases. MethodsTo overcome these challenges, we developed methods to broaden FVTs clinical application while maintaining efficacy and increasing safety. Specifically, we employed the following approaches: 1) Chemostat-fermentation to reproduce the bacteriophage FVT donor component and remove eukaryotic viruses (FVT-ChP), 2) solvent-detergent treatment to inactivate enveloped viruses (FVT-SDT), and 3) pyronin-Y treatment to inhibit RNA-virus replication (FVT-PyT). We assessed the efficacy of these processed FVTs in a C. difficile infection mouse model and compared them with untreated FVT (FVT-UnT), FMT, and saline. ResultsFVT-SDT, FVT-UnT, and FVT-ChP reduced the incidence of mice reaching the humane endpoint (0/8, 2/7, and 3/8, respectively) compared to the FMT, FVT-PyT, and saline control (5/8, 7/8, and 5/7, respectively) and significantly reduced the load of colonizing C. difficile cells and toxin A/B levels. There was a potential elimination of C. difficile colonization, with 7 out of 8 mice treated with FVT-SDT testing negative with qPCR. In contrast, all other treatments exhibited the continued presence of C. difficile. Moreover, the results were supported by changes in the gut microbiome profiles, cecal cytokine levels and histopathological findings. Assessment of viral engraftment following FMT/FVT treatment and host-phage correlations analysis suggested that transfer of phages likely were an important contributing factor associated with treatment efficacy. ConclusionsThis proof-of-concept study show that specific modifications to FVT hold promise in addressing challenges related to donor variability and infection risks. Two strategies lead to treatments significantly limiting C. difficile colonization in mice, with solvent/detergent treatment and chemostat-propagation emerging as promising approaches.

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Rasmussen, T. S., Forster, S., Larsen, S. B., von Munchow, A., Tranaes, K. D., Brunse, A., Castro-Mejia, J. L., Adamberg, S., Hansen, A. K., Adamberg, K., Hansen, C. H. F., Nielsen, D. S.. 2023-03-17. Development of safe and effective bacteriophage-mediated therapies against C. difficile infections a proof-of-concept preclinical study. https://doi.org/10.1101/2023.03.17.532897

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