Venomics and antivenomics from the ADDovenom project, towards a new generation of antivenoms based on virus like-particles.

Snakebite, a neglected tropical disease, annually accounts for over 120,000 fatalities and significantly exacerbates disability rates. While antivenom, sourced from hyperimmunized animal serum, remains the conventional therapeutic approach, its efficacy is inconsistent, often accompanied by adverse effects, and production costs are prohibitively high. Addressing these challenges, the multidisciplinary European initiative, ADDovenom, seeks to revolutionize snakebite treatment through the implementation of ADDomer©, a thermally stable synthetic virus-like particle with high toxin affinity, engineered for swift toxin clearance post-envenomation. Employing advanced mass spectrometry and proteomics, the ADDovenom project aims to catalog the toxin profiles of nine highly venomous snakes in sub-Saharan Africa, focusing on five Dendroapis and four Echis species. Proteomic analyses utilizing the innovative Multi-Enzymatic Limited Digestion (MELD) technique elucidated venom compositions, revealing distinct molecular weight distributions between Dendroaspis and Echis species. Notably, Dendroaspis species predominantly comprised large peptides within the 6-15 kDa range, while Echis species exhibited higher molecular weight proteins (up to 120 kDa). Analysis indicated that approximately 46% of Dendroaspis venoms comprised toxins, with 3-finger toxins, kunitz-type toxins, and snake venom metalloproteinases as prevalent classes. Echis venoms contained nearly 49% toxins and proteases, with snake venom metalloproteinases, C-type lectin-like proteins, and serine proteinases being highly expressed. Common peptide constituents were identified among Dendroaspis and Echis species, facilitating subsequent bioinformatics modeling for toxin structure determination. Additionally, a preliminary assessment of antivenom potency was proposed, employing immunocapture techniques with antivenom antibodies tethered to magnetic beads. This method promises enhanced understanding of antivenom mechanisms and efficacy, while enabling comparative analyses with innovative ADDobodies/ADDomers constructs. Furthermore, compared to actual antivenomics approach, using microcolumn, the use of magnetic beads and shotgun proteomics allow to decrease significantly the amount of antivenom and venom needed for such experiment and can be automated in 96 well plate.