Periodic Reporting for period 3 - ADDovenom (ADDovenom: Novel Snakebite Therapy Platform of Unparalleled Efficacy, Safety and Affordability)
Periodo di rendicontazione: 2023-04-01 al 2025-03-31
Antivenom is the first-choice treatment of snakebite envenoming (SBE) since 1890 when the first antivenoms were developed. Nevertheless, this Neglected Tropical Disease still, annually, causes up to 138,000 deaths and 400,000 amputations and other permanent disabilities in surviving victims. This disease burden primarily affects the most economically and medically disadvantaged farming communities of Asia, sub-Saharan Africa and Latin America. Children often suffer more severe effects than adults, due to their smaller body mass. Current anti-venoms (AVs) are based on antibodies from hyperimmunised horses and sheep. These AVs are weakly effective: only 10-15% of the IgGs bind venom and even fewer neutralise venom toxins, and multiple vials are needed to effect cure, but each additional vial induces higher levels of adverse effects and increases treatment costs. An additional disadvantage of current AVs is that they cannot rationally incorporate the distinct immunogenicity or toxicity of the venoms’ proteins into the design (venoms comprise between 20 to >100 proteins that vary in molecular mass, bioactivity and pathogenicity). This is particularly problematic because snake venoms and their toxins vary significantly at every taxonomic level, and highly pathogenic, diverse and low-molecular mass toxins are notoriously poor immunogens. Our ambition is to generate a safe, affordable and efficient antivenom for Sub-Saharan Africa using innovative synthetic biology, biochemical and proteomics approaches. This aligns with WHO’s strategy to halve snakebite mortality and morbidity by 2030.
The ADDovenom consortia have generated neutralising ADDobodies/ADDomers or nanobodies against pathogenic snake toxins as we set out in the ADDovenom work programme. We are excited and confident that our results provide a basis for completely synthetic antivenoms to protect against the medically most relevant venomous snakes in Sub-Saharan Africa.
The ADDovenom consortia have generated neutralising ADDobodies/ADDomers or nanobodies against pathogenic snake toxins as we set out in the ADDovenom work programme. We are excited and confident that our results provide a basis for completely synthetic antivenoms to protect against the medically most relevant venomous snakes in Sub-Saharan Africa.
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.
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.
Progress beyond the state of the art:
Bristol has developed an efficient protocol to produce and purify active SVMPs using recombinant technology. The corresponding publication, to be submitted very soon, is appended to the RP3 report.
ADDobodies have been validated for excellent expression yields and thermostability. A paper describing the structure and thermostability of the ADDobodies has been published (Buzas et al. 2024).
LSTM and Liège have completed their analysis of the Dendroaspis polylepis and Echis ocellatus venoms. A publication has been submitted.
A novel magnetic bead based method to analyze snake venom has been developed and validated by Liege.
Bristol and Marseille have developed amn efficient protocol to purify at high yields ADDomer-based antivenoms, this will be part of an upcoming piblication prepared by the consortium.
Neutrazation of recombinant PLA2 toxins and whole venom by ADDobodies and ADDomer antitoxins has been demonstrated by the ADDovenom consortium (led by Bristol and LSTM), a corresponding publication is in preparation.
Up-scaling of ADDOmer-based antivenom production has been deomnstrated by iBET.
Expected results until the end of Project:
We have generated neutralising ADDobodies/ADDomers or nanobodies against pathogenic snake toxins as we set out in the ADDovenom work programme. We are excited and confident that our results provide a basis for completely synthetic antivenoms to protect against the medically most relevant venomous snakes in Sub-Saharan Africa.
Impacts:
New collaborative projects emerged within the ADDovenom consortium which already now led to additional funding:
A Wellcome Trust Collaborator Award supports the establishment of additional international academic (Nigeria, India) and private company (IAVI, USA) collaborations and the establishment of long-term collaborations, and acceleration of ADDovenom impacts
A UK Research and Innovation Engineering Biology Mission Award: Haemotoxic and cytotoxic snake venom metalloproteinases - production, enzymatic specificity, snakebite treatment, and biomedical use - will further support ADDovenom innovations
The ADDovenom consortium (all five partners) have submitted an application for an MSCA Doctoral Network: SNAKEBITN. This draws in additional experts 1) Prof. Gerard Lambeau, CNRS, and Chair of our ADDovenom Scientific Advisory Board; (2) Prof. Timothy Jenkins, Centre for Antibody technology at TU Copenhagen (Denmark); (3) Prof Jan Tytgat, KU Leuven; and (4) Prof. George Oluoch, is the Head of the Kenyan Snakebite Research and Interventions Centre (K-SRIC), a Kenyan Ministry of Health research institute.
LSTM, Bristol, Marseille and Liege are actively participating in the European Venom Network (EUVEN), EU COST Action (CA19144) which aims at consolidating collaboration and knowledge exchange between European researchers working on venoms creating additional synergies.
Bristol has developed an efficient protocol to produce and purify active SVMPs using recombinant technology. The corresponding publication, to be submitted very soon, is appended to the RP3 report.
ADDobodies have been validated for excellent expression yields and thermostability. A paper describing the structure and thermostability of the ADDobodies has been published (Buzas et al. 2024).
LSTM and Liège have completed their analysis of the Dendroaspis polylepis and Echis ocellatus venoms. A publication has been submitted.
A novel magnetic bead based method to analyze snake venom has been developed and validated by Liege.
Bristol and Marseille have developed amn efficient protocol to purify at high yields ADDomer-based antivenoms, this will be part of an upcoming piblication prepared by the consortium.
Neutrazation of recombinant PLA2 toxins and whole venom by ADDobodies and ADDomer antitoxins has been demonstrated by the ADDovenom consortium (led by Bristol and LSTM), a corresponding publication is in preparation.
Up-scaling of ADDOmer-based antivenom production has been deomnstrated by iBET.
Expected results until the end of Project:
We have generated neutralising ADDobodies/ADDomers or nanobodies against pathogenic snake toxins as we set out in the ADDovenom work programme. We are excited and confident that our results provide a basis for completely synthetic antivenoms to protect against the medically most relevant venomous snakes in Sub-Saharan Africa.
Impacts:
New collaborative projects emerged within the ADDovenom consortium which already now led to additional funding:
A Wellcome Trust Collaborator Award supports the establishment of additional international academic (Nigeria, India) and private company (IAVI, USA) collaborations and the establishment of long-term collaborations, and acceleration of ADDovenom impacts
A UK Research and Innovation Engineering Biology Mission Award: Haemotoxic and cytotoxic snake venom metalloproteinases - production, enzymatic specificity, snakebite treatment, and biomedical use - will further support ADDovenom innovations
The ADDovenom consortium (all five partners) have submitted an application for an MSCA Doctoral Network: SNAKEBITN. This draws in additional experts 1) Prof. Gerard Lambeau, CNRS, and Chair of our ADDovenom Scientific Advisory Board; (2) Prof. Timothy Jenkins, Centre for Antibody technology at TU Copenhagen (Denmark); (3) Prof Jan Tytgat, KU Leuven; and (4) Prof. George Oluoch, is the Head of the Kenyan Snakebite Research and Interventions Centre (K-SRIC), a Kenyan Ministry of Health research institute.
LSTM, Bristol, Marseille and Liege are actively participating in the European Venom Network (EUVEN), EU COST Action (CA19144) which aims at consolidating collaboration and knowledge exchange between European researchers working on venoms creating additional synergies.