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AAV-mediated delivery of actoxumab and bezlotoxumab results in serum and mucosal antibody concentrations that provide protection from C. difficile toxin challenge

Gene Therapy volume 30, pages 455–462 (2023)Cite this article

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Abstract

Clostridium difficile is the leading cause of antibiotic-associated nosocomial diarrhea in the developed world. When the host-associated colon microbiome is disrupted by the ingestion of antibiotics, C. difficile spores can germinate, resulting in infection. C. difficile secretes enterotoxin A (TcdA) and cytotoxin B (TcdB) that are responsible for disease pathology. Treatment options are limited as the bacterium demonstrates resistance to many antibiotics, and even with antibacterial therapies, recurrences of C. difficile are common. Actotoxumab and bezlotoxumab are human monoclonal antibodies that bind and neutralize TcdA and TcdB, respectively. In 2016, the US food and drug administration (FDA) approved bezlotoxumab for use in the prevention of C. difficile infection recurrence. To ensure the long-term expression of antibodies, gene therapy can be used. Here, adeno-associated virus (AAV)6.2FF, a novel triple mutant of AAV6, was engineered to express either actotoxumab or bezlotoxumab in mice and hamsters. Both antibodies expressed at greater than 90 μg/mL in the serum and were detected at mucosal surfaces in both models. Hundred percent of mice given AAV6.2FF-actoxumab survived a lethal dose of TcdA. This proof of concept study demonstrates that AAV-mediated expression of C. difficile toxin antibodies is a viable approach for the prevention of recurrent C. difficile infections.

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Fig. 1: Actoxumab and Bezlotoxumab serum expression.
Fig. 2: Actoxumab and bezlotoxumab mucosal surface expression.
Fig. 3: Toxin binding activity of AAV-expressed actoxumab and bezlotoxumab in serum.
Fig. 4: Bezlotoxumab serum and mucosal surface expression in Syrian hamsters.
Fig. 5: AAV6.2FF-mediated expression of actoxumab extends survival in the murine TcdA challenge model.
Fig. 6: AAV6.2FF-mediated expression of bezlotoxumab does not extend survival in the murine TcdB challenge model.

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Acknowledgements

We thank all those involved in the care of the animals for these studies at all institutions.

Funding

This research was funded by NSERC Discovery Grant number RGPIN-2018-04737. BAYS and ADR are both recipients of an OVC Scholarship and an Ontario Graduate Scholarship.

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Author notes

  1. These authors contributed equally: Matthew M. Guilleman, Brenna A.Y. Stevens

Authors and Affiliations

  1. Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada

    Matthew M. Guilleman, Brenna A. Y. Stevens, Laura P. Van Lieshout, Amira D. Rghei, Yanlong Pei, Lisa A. Santry & Sarah K. Wootton

  2. Avamab Pharma Inc., Calgary, AB, Canada

    Matthew M. Guilleman & Brad Thompson

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Correspondence to Sarah K. Wootton.

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Conflict of interest

LPvL and SKW are inventors on a US patent for the AAV6.2FF capsid. This patent (US20190216949) is licensed to Avamab Pharma Inc., where BT and SKW are co-founders and BT serves as an executive.

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Guilleman, M.M., Stevens, B.A.Y., Van Lieshout, L.P. et al. AAV-mediated delivery of actoxumab and bezlotoxumab results in serum and mucosal antibody concentrations that provide protection from C. difficile toxin challenge. Gene Ther 30, 455–462 (2023). https://doi.org/10.1038/s41434-021-00236-y

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