Development of Galleria mellonella as a replacement preclinical model for snakebite envenoming to reduce the need for murine models.

Snakebite envenoming (SBE) annually kills ~100,000 people worldwide, with 4-fold more suffering life-altering morbidity. Antivenom, the only approved treatment, requires preclinical models to assess efficacy and safety as an essential part of the regulated manufacturing process. In accordance with the World Health Organization (WHO) 'Guidelines for the production, control, and regulation of antivenom immunoglobulins', the only validated assays to determine the potential clinical effectiveness of an antivenom are preclinical rodent models (typically mice).

These procedures are rated 'severe' in the UK and EU, are labour-intensive and require high animal numbers. Following the inclusion of SBE on the WHO Neglected Tropical Disease (NTD) list in 2017, supported by WHO's SBE 2019 roadmap, there has been increased research into SBE therapies, focused on improved efficacy, safety and affordability. The increased activity in this area further emphasises the need for a validated, alternative model to the current severe models, to minimise the suffering of thousands of mice per year.

Galleria mellonella (herein referred to as Galleria) have gained momentum as a replacement for vertebrate models in the last decade. Developing a Galleria model for SBE would provide a lower-cost, lower-labour intensive, high-throughput non-vertebrate alternative model.

Our pilot data demonstrates that Galleria envenoming pathology is comparable to mammalian envenoming - i.e. coagulopathic venoms impair the clotting function of both mammalian blood and Galleria hemolymph, and neurotoxic venoms cause rapid paralysis in mammals and Galleria. In this project, we aim to validate a replacement model to reduce the numbers of mice in SBE research and antivenom manufacture through three work packages.

In work package 1 we will enhance our prototype live/dead Galleria model to an unbiased model of venom-induced neurotoxicity, coagulopathy and tissue damage. In work package 2, we will assess if the enhanced Galleria model is fit-for-purpose as a viable alternative invertebrate in vivo model for SBE. This will be achieved through direct comparisons of the LD50s (median lethal dose of venom) and ED50s (median effective dose of an antivenom) between Galleria and existing murine data, for a panel of medically important venoms and antivenoms.

Work package 3 will focus on promoting this model as an alternative to the severe-rated SBE mouse model through; i) incorporation of this model into routine practice by an industrial antivenom manufacturer (ICP) and in academic settings (LSTM, UoL and LU), and ii) a clear dissemination strategy including publications, conference presentations, international workshops and curation of a permanent website.