Within the third reporting period of ADDovenom we have submitted all remaining deliverables, and have successfully reached nine milestones.
For mass spectrometric and bioinformatics characterisation of venoms (WP2) final refinements were made to the toxin sequences of nine snake venoms, achieving the completion and formatting of the final ADDovenom database (D2.7). Additionally, a comprehensive dataset was compiled, including venom component sequences, post-translational modifications (PTMs), potential mutations, and isoelectric points. With D2.4 and D2.6 completed ahead of schedule, two innovative research directions were pursued to deepen insights into venom composition, envenomation mechanisms, and antivenom strategies: 1) Magnetic Bead-Based Antivenomic Strategy and 2) MALDI Mass Spectrometry Imaging for Venom Action Analysis.
In Bristol and Marseille, we characterised selected anti-phospholipase A2 (PLA2) binding ADDobodies, determined neutralising IC50 values, and established potential for broadly neutralisaing potential agains PLA2s from other viper species. Three ADDobodies were converted into the ADDomer format; Of these, ADDomer C5 produced soluble, correctly assembled nanoparticles, for which we have established neutralising IC50 value against recombinant PLA2s and Echis coloratus venom.
We have achieved a breakthrough in establishing a generic protocol for recombinant production of cytotoxic and haemotoxic snake venom metalloproteinases (SVMPs).
In WP4, LSTM have established and refined toxin activity assays for use in this project. recombinant neurotoxins produced in WP3 and purified venom fractions from native mamba venoms have also been tested in these assays. ADDobody testing has commenced, with lead anti-PLA2 ADDobodies entering inhibition testing in PLA2 assays. A ‘bridge assay’ seeking to reduce the long term ethical costs of murine in vivo experiments and simultaneously increase the throughput of inhibitor testing has also been developed, using chicken embryos as an insensate in vivo model.
In WP5 iBET have focused on finalizing the downstream process development for the non-functionalized ADDomer (Task 5.3) and applying the processes developed for the non-functionalized ADDomer were applied to the production and purification of functionalized ADDomer C5. Purified functionalized ADDomer C5 particles had >95% purity, with a yield of 33 mg per L of culture.
All partners engaged in Communication, Dissemination and Exploitation Activities (WP6). The ADDovenom Dissemination and Exploitation Report highlights the impacts to date.
