Venomics and antivenomics from the European ADDovenom project, towards a new generation of antivenoms based on Virus Like-Particules.

Snakebite is a neglected tropical disease causing more than 120,000 deaths a year and 3-4
times as many disabilities. Antivenom, obtained from the serum of hyperimmunised
animals, remains the standard treatment. Nevertheless, antivenoms are not always
effective, have undesirable side effects and are expensive to produce. ADDovenom, a
multidisciplinary European project, aims to bring about a rapid change in the treatment of
snakebites using ADDomer©, a thermostable synthetic virus-like particle with high affinity,
designed to rapidly eliminate deleterious toxins from the bloodstream after envenomation.
The ADDovenom project is exploiting the potential of advanced mass spectrometry and
proteomics to inventory the toxin repertoire of nine of the most deadly snakes in sub Saharan Africa, in order to analyse the effectiveness of the ADDomer technology. 5 species
of Dendroapis and 4 species of Echis were selected. Proteomics of the venoms (10 ug of
each) was carried out using an innovative methodology named Multi-Enzymatic Limited
Digestion - MELD. Venoms were analysed using an Acquity M-Class coupled to a Q Exactive™ mass spectrometer (Top 12). Protein identification and relative quantification
were performed by Peaks Studio X+, using the venom gland transcriptomes. The nine
venoms contain compounds with a wide range of molecular weights, mainly from 1 to 120
kDa. Dendroaspis species had a majority of large peptides in the 6-15 kDa range, while
Echis species were found to be rich in higher molecular weight proteins (up to 120 kDa).
Proteomics results showed that around 46% of Dendroaspis venoms were composed of
toxins, with 3-finger toxins, kunitz-type toxins and snake venom metalloproteinase being
the most highly expressed toxin classes. Echis contained almost 49% of toxins and
proteases, with snake venom metalloproteinases, C-type lectin-like proteins and serine
proteinases being the most expressed. 62 peptides were found in common among
Dendroaspis species and 103 peptides were shared in all Echis species. These antigens can
be subjected to bioinformatics modelling (Alphafold) to identify their position in the 3D
structure of complete toxins. In this study, a preliminary assessment of the potency of
antivenoms is also proposed. This method involves the immunocapture of venom toxins by
antivenom antibodies grafted onto magnetic beads. This approach will not only provide a
better understanding of the mode of action and efficacy of various antivenoms, but will also
enable them to be compared with our innovative Addobodies/Addomers constructs.